U.S. patent application number 12/528828 was filed with the patent office on 2013-07-11 for protein production.
This patent application is currently assigned to BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG. The applicant listed for this patent is Eric Becker, Lore Florin, Tim Fugmann, Angelika Hausser, Hitto Kaufmann, Monilola Olayioye. Invention is credited to Eric Becker, Lore Florin, Tim Fugmann, Angelika Hausser, Hitto Kaufmann, Monilola Olayioye.
Application Number | 20130177919 12/528828 |
Document ID | / |
Family ID | 39639553 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177919 |
Kind Code |
A1 |
Kaufmann; Hitto ; et
al. |
July 11, 2013 |
PROTEIN PRODUCTION
Abstract
The invention concerns the field of protein production and cell
culture technology. CERT is identified as a novel in vivo PKD
substrate. Phosphorylation on serine 132 by PKD decreases the
affinity of CERT towards its lipid target phosphatidylinositol
4-phosphate at Golgi membranes and reduces ceramide transfer
activity, identifying PKD as a regulator of lipid homeostasis. The
present invention shows that CERT in turn is critical for PKD
activation and PKD dependent protein cargo transport to the plasma
membrane. The interdependence of PKD and CERT is thus a key to the
maintenance of Golgi membrane integrity and secretory
transport.
Inventors: |
Kaufmann; Hitto; (Ulm,
DE) ; Florin; Lore; (Biberach, DE) ; Becker;
Eric; (Hochdorf, DE) ; Olayioye; Monilola;
(Ulm, DE) ; Hausser; Angelika; (Stuttgart, DE)
; Fugmann; Tim; (Zuerich, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kaufmann; Hitto
Florin; Lore
Becker; Eric
Olayioye; Monilola
Hausser; Angelika
Fugmann; Tim |
Ulm
Biberach
Hochdorf
Ulm
Stuttgart
Zuerich |
|
DE
DE
DE
DE
DE
CH |
|
|
Assignee: |
BOEHRINGER INGELHEIM PHARMA GMBH
& CO. KG
Ingelheim
DE
|
Family ID: |
39639553 |
Appl. No.: |
12/528828 |
Filed: |
February 29, 2008 |
PCT Filed: |
February 29, 2008 |
PCT NO: |
PCT/EP2008/052493 |
371 Date: |
March 12, 2010 |
Current U.S.
Class: |
435/6.13 ;
435/194; 435/320.1; 435/328; 435/358; 435/361; 435/365; 435/369;
435/455; 435/69.1; 435/69.6; 435/7.21; 435/7.92; 530/324; 530/363;
530/387.3 |
Current CPC
Class: |
A61P 35/04 20180101;
C12P 21/00 20130101; C07K 14/4702 20130101; C07K 2319/036 20130101;
C07K 16/00 20130101; A61P 31/00 20180101; C07K 14/435 20130101;
A61P 43/00 20180101; A61P 35/00 20180101; C12Q 1/68 20130101; C07K
14/47 20130101; C12N 15/63 20130101; C12N 15/67 20130101; G01N
33/68 20130101; C12N 15/85 20130101 |
Class at
Publication: |
435/6.13 ;
530/387.3; 530/324; 530/363; 435/69.1; 435/69.6; 435/455;
435/320.1; 435/7.92; 435/7.21; 435/328; 435/358; 435/361; 435/194;
435/369; 435/365 |
International
Class: |
C12N 15/85 20060101
C12N015/85; G01N 33/68 20060101 G01N033/68; C12Q 1/68 20060101
C12Q001/68; C07K 14/47 20060101 C07K014/47; C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 2, 2007 |
EP |
07103406.0 |
Mar 15, 2007 |
EP |
07104226.1 |
Sep 13, 2007 |
EP |
07116358.8 |
Claims
1. Method of producing a heterologous protein of interest in a cell
comprising a. Increasing the expression or activity of a protein
having an amino acid sequence comprising a steroidogenic acute
regulatory related lipid transfer (START) domain or a derivative or
mutant thereof, and b. Effecting the expression of said protein of
interest.
2. Method according to claim 1 whereby the START domain protein is
a mammalian START domain family member such as PCTP (SEQ ID NO.
27), StarD7, GPBP, StarD10, StarD8, StarD13, DLC-1, StarD4 (SEQ ID
NO. 21), StarD6 (SEQ ID NO. 25), StarD5 (SEQ ID NO. 23), MLN64,
StAR, THEA-2, CACH or StarD9 or a derivative or mutant thereof.
3. Method according to claim 1 whereby the START domain protein is
characterized by being induced upon ER stress and/or is
structurally characterized by consisting solely of a START domain
such as StarD4 (SEQ ID NO. 21), StarD5 (SEQ ID NO. 23), StarD6 (SEQ
ID NO. 25) or phosphatidylcholin transfer protein (PCTP) (SEQ ID
NO. 27).
4. Method according to claim 1 whereby the START domain comprises
at least the START domain consensus sequence (SEQ ID NO 28), or at
least the 219 amino acid START domain of CERT.sub.L (SEQ ID NO.
19), or at least the 223 amino acid START domain of CERT and CERT
S132A (SEQ ID NO. 17), or at least the START domain of StarD4 (SEQ
ID NO. 21) or at least the START domain of StarD5 (SEQ ID NO. 23)
or a derivative or mutant thereof.
5. Method according to claim 1 whereby the START domain protein is
ceramide transfer protein CERT (SEQ ID NO. 11 or SEQ ID NO. 13) or
a derivative or mutant thereof.
6. Method according to claim 5 whereby the START domain protein is
mutated ceramide transfer protein CERT and said mutation disables
and/or deletes a phosphorylation site at any serine, threonine or
tyrosine position of CERT.
7. Method according to claim 6 whereby the START domain protein is
mutated ceramide transfer protein CERT and said mutation disables
and/or deletes the protein kinase D (PKD) phosphorylation site of
CERT at position 132.
8. Method according to claim 7 whereby the mutated CERT is CERT
S132A (SEQ ID NO. 15).
9. Method according to claim 1 whereby said method results in
increased specific cellular productivity of said protein of
interest in said cell in comparison to a control cell expressing
said protein of interest, but whereby said control cell does not
have increased expression or activity of a protein having an amino
acid sequence comprising a steroidogenic acute regulatory related
lipid transfer (START) domain or a derivative or mutant
thereof.
10. Method according to claim 9 whereby the increase in
productivity is about 5% to about 10%, about 11% to about 20%,
about 21% to about 30%, about 31% to about 40%, about 41% to about
50%, about 51% to about 60%, about 61% to about 70%, about 71% to
about 80%, about 81% to about 90%, about 91% to about 100%, about
101% to about 149%, about 150% to about 199%, about 200% to about
299%, about 300% to about 499%, or about 500% to about 1000%.
11. Method according to claim 1 whereby said cell is a eukaryotic
cell such as a yeast, plant, worm, insect, avian, fish, reptile or
mammalian cell.
12. Method according to claim 11 whereby said eukaryotic cell is a
mammalian cell.
13. Method according to claim 12 whereby said mammalian cell is a
Chinese Hamster Ovary (CHO), monkey kidney CV1, monkey kidney COS,
human lens epithelium PER.C6.TM., human embryonic kidney, HEK293,
baby hamster kidney, African green monkey kidney, human cervical
carcinoma, canine kidney, buffalo rat liver, human lung, human
liver, mouse mammary tumor or myeloma cell, a dog, pig or macaque
cell, rat, rabbit, cat, goat, preferably a CHO cell.
14. Method according to claim 13 whereby said CHO cell is CHO wild
type, CHO K1, CHO DG44, CHO DUKX-B11, CHO Pro 5.
15. Method according to claim 1 whereby the protein of interest is
a membrane or secreted protein.
16. Method according to claim 15 whereby the protein of interest is
an antibody or antibody fragment.
17. Method according to claim 16 whereby the antibody is
monoclonal, polyclonal, mammalian, murine, chimeric, humanized,
primatized, primate, human or an antibody fragment or derivative
thereof such as antibody, immunoglobulin light chain,
immunoglobulin heavy chain, immunoglobulin light and heavy chains,
Fab, F(ab')2, Fc, Fc-Fc fusion proteins, Fv, single chain Fv,
single domain Fv, tetravalent single chain Fv, disulfide-linked Fv,
domain deleted, minibody, diabody, or a fusion polypeptide of one
of the above fragments with another peptide or polypeptide,
Fc-peptide fusion, Fc-toxine fusion, scaffold proteins.
18. Method of increasing specific cellular productivity of a
membrane or secreted protein of interest in a cell comprising
introducing into a cell one or more vector systems comprising
nucleic acid sequences encoding for at least two polypeptides
whereby a. a first polynucleotide encodes a protein having an amino
acid sequence comprising a steroidogenic acute regulatory related
lipid transfer (START) domain or a derivative or mutant thereof and
b. a second polynucleotide encodes a protein of interest c. and
whereby the protein of interest and the protein having an amino
acid sequence comprising a steroidogenic acute regulatory related
lipid transfer (START) domain or a derivative or mutant thereof are
expressed by said cell.
19. Method of increasing the transfection efficiency of a cell
expressing a membrane or secreted protein of interest in a cell
comprising a. transfecting said cell with a first polynucleotide
encoding a protein having an amino acid sequence comprising a
steroidogenic acute regulatory related lipid transfer (START)
domain or a derivative or mutant thereof, b. subsequently
transfecting said cell with a second polynucleotide encoding a
protein of interest, c. whereby said first and second
polynucleotides are located on different vector systems.
20. Expression vector comprising two polynucleotides, a. a first
polynucleotide encoding for a protein having an amino acid sequence
comprising a steroidogenic acute regulatory related lipid transfer
(START) domain or a derivative or mutant thereof and b. a second
polynucleotide encoding for a protein of interest.
21. Expression vector according to claim 20 whereby the START
domain protein is a mammalian START domain family member such as
PCTP, StarD7, GPBP, StarD10, StarD8, StarD13, DLC-1, StarD4,
StarD6, StarD5, MLN64, StAR, THEA-2, CACH or StarD9 or a derivative
or mutant thereof.
22. Expression vector according to claim 20 whereby the START
domain protein is ceramide transfer protein CERT (SEQ ID NO. 11 or
SEQ ID NO. 13) or a derivative or mutant thereof.
23. Expression vector according to claim 22 whereby the mutated
CERT is CERT.sub.S132A (SEQ ID NO. 15).
24. Expression vector of claim 20 whereby said first polynucleotide
increases the protein transport in a cell via the secretory
pathway.
25. A cell comprising the expression vector of claim 20.
26. A cell according to claim 25 whereby said cell is a eukaryotic
cell such as a yeast, plant, worm, insect, avian, fish, reptile or
mammalian cell.
27. A cell according to claim 26 whereby said eukaryotic cell is a
mammalian cell.
28. A cell according to claim 27 whereby said mammalian cell is a
Chinese Hamster Ovary (CHO), monkey kidney CV1, monkey kidney COS,
human lens epithelium PER.C6.TM., human embryonic kidney, HEK 293,
baby hamster kidney, African green monkey kidney, human cervical
carcinoma, canine kidney, buffalo rat liver, human lung, human
liver, mouse mammary tumor or myeloma cell, a dog, pig or macaque
cell, rat, rabbit, cat, goat.
29. A cell according to claim 28 whereby said CHO cell is CHO wild
type, CHO K1, CHO DG44, CHO DUKX-B11, CHO Pro-5.
30. A protein of interest produced by the method of claim 1.
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. Method for identifying a modulator of START domain protein
function comprising a. providing a protein having an amino acid
sequence comprising a steroidogenic acute regulatory related lipid
transfer (START) domain or a derivative or mutant thereof, b.
contacting said protein of step a) with a test agent, c.
determining an effect related to increased or decreased protein
secretion or expression of cell-surface proteins.
36. (canceled)
37. (canceled)
38. (canceled)
39. Method according to claim 12 whereby said mammalian cell is a
CHO cell.
40. Method according to claim 13 whereby said CHO cell is CHO
DG44.
41. A cell according to claim 27 whereby said mammalian cell is a
CHO cell.
42. A cell according to claim 28 whereby said CHO cell is CHO
DG44.
43. The protein of interest according to claim 30, wherein said
protein is an antibody.
44. Method according to claim 35, wherein said START domain protein
function is CERT function.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Technical Field
[0002] The invention concerns the field of cell culture technology.
It concerns a method for producing proteins as well as a method to
generate novel expression vectors and host cells for
biopharmaceutical manufacturing. The invention further concerns
pharmaceutical compositions and methods of treatment.
[0003] 2. Background
[0004] The market for biopharmaceuticals for use in human therapy
continues to grow at a high rate with 270 new biopharmaceuticals
being evaluated in clinical studies and estimated sales of 30
billions in 2003 (Werner, 2004). Biopharmaceuticals can be produced
from various host cell is systems, including bacterial cells, yeast
cells, insect cells, plant cells and mammalian cells including
human-derived cell lines. Currently, an increasing number of
biopharmaceuticals is produced from eukaryotic cells due to their
ability to correctly process and modify human proteins. Successful
and high yield production of biopharmaceuticals from these cells is
thus crucial and depends highly on the characteristics of the
recombinant monoclonal cell line used in the process. Therefore,
there is an urgent need to generate new host cell systems with
improved properties and to establish methods to culture producer
cell lines with high specific productivities as a basis for high
yield processes.
[0005] Early approaches focused on process design and reactor
design. Now the main improvements are driven by media formulation
development and genetically engineering of host cells. The most
common industrial mammalian host cell systems for the production of
biopharmaceuticals are immortalized Chinese hamster ovary (CHO)
cell lines (Wurm, 2004).
[0006] Initial metabolic engineering strategies to improve
mammalian production cell lines focused on their ability to grow in
suspension in serum free media. Stable expression of transferrin
and insulin-like growth factor 1 (IGF-1) in CHO-K1 cells resulted
in a cell line able to proliferate under protein-free conditions
(Pak et al., 1996). Further approaches to improve the production
cell lines included the use of regulatory DNA elements on the
transfection vectors aimed to target or create transcriptional hot
spots. Regulatory elements such as S/MARs
(Scaffold/matrix-associated regions) which effect chromatin
structure and UCOEs (Ubiquitous chromatin opening elements) derived
from house keeping genes were both shown to positively effect
specific productivities of recombinant proteins produced from CHO
cell lines (Barnes and Dickson, 2006).
[0007] As apoptosis has been shown to be the predominant cause of
cell death in mammalian cell culture production processes
(al-Rubeai and Singh, 1998) the effect of expression of
anti-apoptotic genes in mammalian host cells on culture viability
was thoroughly investigated. Most antiapoptosis engineering
strategies are focused on the overexpression of anti-apoptotic
genes of the bcl-2 family (e.g. bcl-1 or bcl-xL; (Kaufmann and
Fussenegger, 2003). By increasing the cellular resistance to
apoptotic stimuli during fermentation, such as nutrient depletion
and waste byproduct accumulation, production processes with
apoptosis engineered cell lines showed prolonged culture viability
and in some cases an increase in product yield (Chiang and Sisk,
2005).
[0008] Since most biopharmaceutical products are proteins that are
secreted from the cells during the production process, the
secretory transport machinery of the production cell line is
another interesting target for novel host cell engineering
strategies.
[0009] Protein secretion is a complex multi-step mechanism:
Proteins destined to be transported to the extracellular space or
the outer plasma membrane are first co-translationally imported
into the endoplasmic reticulum. From there, they are packed in
lipid vesicles and transported to the Golgi apparatus and finally
from the trans-Golgi network (TGN) to the plasma membrane where
they are released into the culture medium (Seth et al., 2006).
[0010] The yield of any biopharmaceutical production process
depends largely on the amount of protein product that the producing
cells secrete per time when grown under process conditions. Many
complex biochemical intracellular processes are necessary to
synthesize and secrete a therapeutic protein from a eukaryotic
cell. All these steps such as transcription, RNA transport,
translation, post-translational modification and protein transport
are tightly regulated in the wild-type host cell line and will
impact on the specific productivity of any producer cell line
derived from this host.
[0011] Many engineering approaches have employed the growing
understanding of the molecular networks that drive processes such
as transcription and translation to increase the yield of these
steps in protein production. However, as for any multi-step
production process, widening a bottle-neck during early steps of
the process chain possibly creates bottle necks further downstream,
especially post translation. Up to a certain threshold, the
specific productivity of a production cell has been reported to
correlate linearly with the level of product gene transcription
(Barnes et al., 2007). Further enhancement of product expression at
the mRNA level, however, may lead to an overload of the protein
synthesis, folding or transport machinery, resulting in
intracellular accumulation of the protein product. Indeed, this can
be frequently observed in current manufacturing processes (FIG.
1).
[0012] Specific targeted engineering approaches aimed to address
this problem and to efficiently improve the secretion of protein
products from eukaryotic cells are hampered by the current lack of
understanding of the complex regulatory network that drives the
transport of proteins to the plasma membrane.
[0013] The first studies on engineering the intracellular transport
of secreted therapeutic proteins were centered around the
overexpression of molecular chaperones like binding protein
BiP/GRP78, protein disulfide isomerase (PDI). Chaperones are
cellular proteins hosted within the endoplasmic reticulum (ER) and
assist the folding and assembly of newly synthesised proteins. In
contrast to what could be expected, BiP overexpression in mammalian
cells has been shown to reduce rather than increase the secretion
of proteins it associates with (Dorner and Kaufman, 1994).
Likewise, PDI overexpression in CHO cells reduced the expression of
a TNFR:FC fusion protein (Davis et al., 2000), whereas the specific
production rate of an antibody was increased by 40% (Borth et al.,
2005). A possible explanation for these surprising findings, that
the increase is of the cell's protein folding capacity creates a
production bottle neck further downstream, is supported by a report
describing ER to cis-Golgi transport problems for IFN-gamma
production in a CHO cell line (Hooker et al., 1999).
[0014] Another recent approach to increase the secretion capacity
of mammalian cells is the heterologous overexpression of the
transcription factor X-box binding protein 1 (XBP-1). XBP-1 is one
of the master-regulators in the differentiation of plasma cells, a
specialized cell type optimized for high-level production and
secretion of antibodies (Iwakoshi et al., 2003). XBP-1 regulates
this process by binding to the so called ER stress responsive
elements (ERSE) within the promoters of a wide spectrum of
secretory pathway genes, resulting in (i) a physical expansion of
the ER, (ii) increased mitochondrial mass and function, (iii)
larger cell size and (iv) enhanced total protein synthesis (Shaffer
et al., 2004).
[0015] Recently, attempts were described to increase protein
secretion by overexpressing XBP-1 in non-plasma cells, especially
production cell lines. In CHO-K1 cells, the production level of two
reporter proteins (secreted alkaline phospatase (SEAP) and secreted
alpha-amylase (SAMY)) was shown to increase after XBP-1
introduction in CHO-K1 cells. However, no effect could be
demonstrated in transient studies with other cell lines such as
HEK293, HeLa or HT-1080 cells (Tigges and Fussenegger, 2006). The
patent application WO2004111194 by Ailor Eric claims the
overexpression of XBP-1 or ATF6 for the generation of highly
productive cell lines.
[0016] Notably, XBP-1 does not only regulate plasma cell
differentiation but also plays an important role in the unfolded
protein response (UPR) (Brewer and Hendershot, 2005). The UPR
represents a complex signal transduction network activated by
inhibition of protein folding in the endoplasmic reticulum (ER).
The UPR coordinates adaptive responses to this stress situation,
including induction of ER resident molecular chaperone and protein
foldase expression to is increase the protein folding capacity of
the ER, induction of phospholipid synthesis, attenuation of general
translation, and upregulation of ER-associated degradation to
decrease the unfolded protein load of the ER. Upon severe or
prolonged ER stress, the UPR ultimately induces apoptotic cell
death (Schroder, 2006).
[0017] The process of terminal differentiation, such as the
maturation from a lymphocyte to a plasma cell, is usually regarded
an apoptosis-like program, during which the cell loses its
proliferative capacity to give rise to a terminally differentiated
secretory cell. In fact, nearly all cell types specifically
designed for high-level protein secretion (e.g. glandular cells,
pancreatic beta cells) are terminally differentiated, are not able
to proliferate and have a limited life-span before ultimately
undergoing programmed cell death (Chen-Kiang, 2003). Therefore,
overexpressing XBP-1 as a regulator of both plasma cell
differentiation and UPR, is potentially disadvantageous due to its
inherent risk to inhibit proliferation and/or induce apoptosis.
[0018] Taken together, there is a need for improving the secretory
capacity of host cells for recombinant protein production. This
might even become more important in combination with novel
transcription-enhancing technologies and in high-titer processes in
order to prevent post-translational bottle necks and intracellular
accumulation of the protein product (FIG. 1). However, at present,
there are two major hurdles on the way to targeted manipulation of
the secretory transport machinery: The still limited knowledge
about the underlying regulatory mechanisms and the requirement to
prevent a concomitant growth-inhibitory or apoptotic response of
the producer cell.
[0019] The present invention describes a novel and surprising role
for the ceramide transfer protein CERT in the transport of secreted
proteins to the plasma membrane and furthermore provides a is
method to efficiently improve the production of proteins that are
transported via the secretory pathway from eukaryotic cells.
[0020] CERT (also known as Goodpasture antigen-binding protein) is
a cytosolic protein essential for the non-vesicular delivery of
ceramide from its site of production at the endoplasmic reticulum
(ER) to Golgi membranes, where conversion to sphingomyelin (SM)
takes place (Hanada et al., 2003).
[0021] Two CERT isoforms exist: the more abundantly expressed,
alternatively spliced form missing a 26-amino-acid, serine-rich
region (SEQ ID NO.10, 11) and the full-length 624 amino acid
protein, designated CERT.sub.L (SEQ ID NO.12,13) (Raya et al.,
2000). Both CERT isoforms possess a carboxyterminal steroidogenic
acute regulatory (StAR)-related lipid transfer (START) domain that
is necessary and sufficient for ceramide binding and transport
(Hanada et al., 2003). START domains are highly conserved from fly
and worm to humans (FIG. 2). They are .about.210 amino acids in
length and form a hydrophobic tunnel that accommodates a monomeric
lipid (Alpy and Tomasetto, 2005; Soccio and Breslow, 2003). START
domains are found in 15 mammalian proteins, with CERT being most
closely related to the phosphatidylcholine transfer protein Pctp,
which binds and shuttles phosphatidylcholine (PC) between
membranes, and StarD10, a lipid transfer protein specific for PC
and PE (Olayioye et al., 2005; Soccio and Breslow, 2003; Wirtz,
2006). In addition to the START domain, the CERT proteins further
contain an aminoterminal PH domain with specificity for PI(4)P that
is responsible for Golgi localization (Hanada et al., 2003; Levine
and Munro, 2002) and a FFAT motif (two phenylalanines in an acidic
tract) that targets the protein to the ER via interaction with the
ER resident transmembrane proteins VAP-A and VAP-B (Kawano et al.,
2006; Loewen et al., 2003).
[0022] The fundamental role of CERT in lipid trafficking was
demonstrated in the Chinese hamster is ovary cell line LY-A, in
which the expression of a mutant non-functional CERT protein
impaired ceramide transport, thus resulting in reduced cellular
levels of sphingomyelin (Hanada et al., 2003). Non-vesicular lipid
transfer is thought to occur at so-called membrane contact sites
(MCS), at which the ER comes into close apposition with other
organelles (Levine and Loewen, 2006). CERT may thus shuttle a very
short distance between ER and Golgi membranes, or perhaps contact
both compartments simultaneously. When overexpressed, the START
domain of CERT is sufficient for ceramide transfer to the Golgi
apparatus (Kawano et al., 2006). However, under physiological
conditions, both Golgi and ER targeting motifs are essential for
CERT function. In LY-A cells, CERT was identified to contain a
mutation within its PH domain (G67E), rendering the protein
defective in PI(4)P binding (Hanada et al., 2003). The requirement
for PI(4)P for CERT function is further supported by a recent
report that PI4KIII-beta activity is necessary for efficient
ceramide trafficking to the Golgi (Toth et al., 2006), the
enzymatic activity of which is stimulated by protein kinase D
(PKD).
[0023] PKD belongs to a subfamily of serine-/threonine-specific
protein kinases (comprising PKD1/PKC.mu., PKD2 and
PKD3/PKC.upsilon.) and was recently identified to be of crucial
importance for the regulation of protein transport from the Golgi
membrane to the plasma membrane (reviewed in (Rykx et al., 2003;
Wang, 2006)). Recruitment and activation of PKD at the TGN is
mediated by the lipid diacylglycerol (DAG; (Baron and Malhotra,
2002)), a pool of which is generated by sphingomyelin synthase from
ceramide and phosphatidylcholine.
[0024] The present invention shows that PKD phosphorylates CERT on
serine 132 adjacent to the PH domain, whereby PI(4)P binding, Golgi
targeting and ceramide transfer activity are negatively regulated.
Furthermore, by transferring ceramide that is required for DAG
production to Golgi membranes, CERT stimulates PKD activity, thus
establishing a regulatory feedback-loop that is ensures the
maintenance of constitutive secretory transport.
[0025] Importantly, the data provided furthermore show that in
different eukaryotic cell lines (COST and HEK293), introduction of
the gene encoding CERT significantly enhances the secretion of a
heterologous protein into the culture medium. This effect is even
more pronounced when using a CERT mutant which cannot be
phosphorylated by PKD. Deletion of the phosphorylation acceptor
site within CERT interrupts the negative control of PKD on CERT,
but leaving the positive feedback of CERT on PKD intact through the
support of ceramide conversion to sphingomyelin and DAG. It can
therefore be speculated that the secretion enhancing mechanism of
the present invention can be exerted not only by wild type CERT but
also by all mutants of CERT which uncouple CERT from the negative
influence of PKD, including point mutations of the acceptor serine,
deletions including this residue as well as mutation or deletion of
the PKD docking site within CERT or even the START domain
alone.
[0026] CERT belongs to the family of StAR-related Lipid Transfer
proteins (Soccio and Breslow, 2003), which are characterized by
their START domains for lipid binding. As the START domain of CERT
has been demonstrated to be both required and sufficient for CERT
action (Hanada et al., 2003), it is possible that the
secretion-promoting effect of CERT could equally be observed when
overexpressing another member of this protein family. This is
especially likely for the closely related members of the
PCTP-subfamily, comprising PCTP (SEQ ID NO.26, 27), CERT/GPBP
itself, StarD7 and StarD10. These proteins have distinct
lipid-binding specificities and could equally impact on the
function of organelles involved in the secretion of heterologous
proteins.
[0027] Furthermore, expression of the related proteins STARD4 (SEQ
ID NO.20, 21) and STARD5 (SEQ ID NO.22, 23), that are induced upon
ER stress, may function to fulfill the increased is demand of lipid
transfer of cells during a production process.
[0028] The existence of START domain proteins in eukaryotic
organisms from fly, worm and mouse to humans indicates that the
basic mechanisms of lipid trafficking are conserved among the
eukaryotic kingdom. It furthermore suggests, that the principle
described in the present invention--that is increasing secretion by
enforced expression of CERT--may well be applicable to all
eukaryotic cells, including yeast.
[0029] In summary, the present invention provides a method for
enhancing the secretory transport of proteins in eukaryotic cells
by heterologous expression of CERT, CERT mutants or another member
of the START protein family. This method is particularly useful for
the generation of optimized host cell systems with enhanced
production capacity for the expression and manufacture of
recombinant protein products.
[0030] The method described in the present invention is
advantageous in several respects:
[0031] First, we demonstrate heterologous expression of CERT to be
a strategy to enhance recombinant protein production by increasing
the secretory capacity of the host cell. Enhancing the specific
productivity of producer cells translates into higher product
yields in industrial protein production processes. With the current
trend towards high-titer processes and more sophisticated
expression enhancing technologies, post-translational bottle necks
will become the evident rate-limiting steps in protein production
and hence will draw increasing attention to secretion engineering
approaches.
[0032] Second, the START domain of CERT is highly conserved in
eukaryotes from C. elegans to humans. This strongly suggests that
the method of the present invention can not only be used in
mammalian host cell systems, but is equally applicable for protein
production in all eukaryotic cells, including insect cells and
yeast cells.
[0033] As a third important feature, CERT as a cytosolic factor is
not part of the unfolded protein response and thus is not involved
in a cellular stress response program which induces the shut-down
of protein translation and--if not resolved--leads to cell cycle
arrest or even apoptosis. In contrast, by playing an independent
role in lipid trafficking, targeting CERT might confer enhanced
protein secretion without concomitant induction of apoptosis. Thus,
overexpressing CERT in producer host cells might be advantageous
over XBP-1 based genetically engineering approaches.
[0034] Fourth, it is shown in the present invention that mutation
of Ser132 of CERT impairs the phosphorylation of CERT by PKD which
frees CERT from a negative regulatory influence. Meanwhile, the
positive stimulation of PKD by CERT via DAG is left intact (FIG.
3A). This finding places CERT in the signalling pathway "upstream"
of PKD, which has been published to be critically involved in the
regulation of the late stages of secretory transport, namely the
transport from the trans-Golgi network to the plasma membrane
(Liljedahl et al., 2001). With regard to protein transport, this
means, that CERT acts "downstream" of the ER which makes CERT the
preferable target for manipulation compared to XBP-1 or specific
ER-residing proteins (FIG. 3B).
[0035] Since CERT can impact even on the latest steps of the
secretory pathway, it can be speculated that heterologous
expression of CERT has the potential to enhance secretion without
creating bottle necks further downstream. To our knowledge, CERT is
currently the most downstream acting target for genetical
engineering of the secretory pathway to enhance heterologous
protein production.
[0036] Taken together, the impact of the lipid-transfer protein
CERT on the secretory transport from ER to Golgi and from the Golgi
apparatus to the plasma membrane, without the disadvantageous
connection to a growth-inhibiting or apoptosis-inducing stress
response make CERT, CERT mutants and other START family proteins
very attractive and promising targets for genetic engineering
approaches aiming to enhance the secretory capacity of eukaryotic
cells.
Applicability
[0037] The targeted manipulation of CERT which is described in the
present invention can be used for a broad range of applications. In
particular, two basic approaches can be distinguished:
(i) Overexpression and/or enhancing the activity of CERT or a CERT
derivative to increase the secretory transport capacity of a cell,
or (ii) reducing CERT activity and/or expression as a means of gene
therapy in order to reduce cancer cell proliferation and/or
invasion.
Applicability of CERT Overexpression
[0038] The described invention describes a method to generate
improved eukaryotic host cells for the production of heterologous
proteins by introducing the gene encoding CERT, CERT mutants or
other proteins of the START protein family. This will enable to
increase the protein yield in production processes based on
eukaryotic cells. It will thereby reduce the cost of goods of such
processes and at the same time reduce the number of batches that
need to be produced to generate the material needed for research
studies, diagnostics, clinical studies or market supply of a
therapeutic protein. The invention will furthermore speed up drug
development as often the generation of sufficient amounts of
material for pre-clinical studies is a critical work package with
regard to the timeline.
[0039] The invention can be used to increase the property of all
eukaryotic cells used for the generation of one or several specific
proteins for either diagnostic purposes, research purposes (target
identification, lead identification, lead optimization) or
manufacturing of therapeutic proteins either on the market or in
clinical development.
[0040] As shown in the present invention, heterologous expression
of CERT does not only enhance protein secretion, but also has an
influence on the abundance of transmembrane proteins on the cell
surface. Inhibition or reduced expression of CERT leads to a
dramatic reduction of the amount of cell surface receptors such as
the transferrin receptor (FIG. 8). As secreted and transmembrane
proteins share the same secretory pathways and are equally
transported in lipid-vesicles, these data underscore the importance
of CERT in the modulation of secretion as well as the transport of
membrane-bound cell-surface receptors.
[0041] Therefore, the method described herein can also be used for
academic and industrial research purposes which aim to characterize
the function of cell-surface receptors. E.g. it can be used for the
production and subsequent purification, crystallization and/or
analysis of surface proteins. This is of crucial importance for the
development of new human drug therapies as cell-surface receptors
are a predominant class of drug targets. Moreover, it might be
advantageous for the study of intracellular signalling complexes
associated with cell-surface receptors or the analysis of
cell-cell-communication which is mediated in part by the
interaction of soluble growth factors with their corresponding
receptors on the same or another cell.
Applicability of Decreasing/Inhibiting CERT
[0042] In the present invention, we provide evidence that the
reduction of CERT expression leads to is reduced secretion of
soluble extracellular proteins as well as a lower abundance of cell
surface receptors. This makes CERT an attractive target for
therapeutic manipulation.
[0043] One of the hallmarks in the conversion from a normal healthy
cell to a cancer cell is the acquisition of independency from the
presence of exogenous growth factors (Hanahan and Weinberg, 2000).
In contrast to the normal cell, tumor cells are able to produce all
growth factors necessary for their survival and proliferation by
themselves. In addition to this autocrine mechanism, cancer cells
often show an upregulated expression of growth factor receptors on
their surface, which leads to an increased responsiveness towards
paracrine-acting growth and survival factors secreted from cells in
the surrounding tissue. By targeting CERT in tumor cells, e.g. by
using siRNA approaches, it might be possible to disrupt autocrine
as well as paracrine growth-stimulatory and/or survival mechanisms
in two ways: (i) By reducing growth factor transport and secretion
and (ii) by decreasing the amount of the corresponding growth
factor-receptor on tumor cells. Thereby both, the amount of growth
stimulating signal and the ability of the cancer cell to perceive
and respond to these signals will be reduced Inhibition of CERT
expression in cancer cells might therefore represent a powerful
tool to prevent cancer cell proliferation and survival.
[0044] CERT might furthermore be a potent therapeutic target to
suppress tumor invasion and metastasis. During the later stages of
most types of human cancer, primary tumors spawn pioneer cells that
move out, invade adjacent tissues, and travel to distant sites
where they may succeed in founding new colonies, known as
metastasis.
[0045] As a prerequisite for tissue invasion, cancer cells express
a whole set of proteases which enable them to migrate through the
surrounding healthy tissue, to cross the basal membrane, to get
into is the blood stream and to finally invade the tissue of
destination. Some of these proteases are expressed as
membrane-bound proteins, e.g. MT-MMPs (Egeblad and Werb, 2002) and
ADAMs (Blobel, 2005). Due to their crucial role in matrix
remodelling, shedding of growth factors and tumor invasion,
proteases themselves are discussed as drug targets for cancer
therapy (Overall and Kleifeld, 2006). We hypothesize that
inhibition of CERT expression and/or activity in tumor cells will
reduce the amount of membrane-bound proteases on the surface of the
targeted cell. This might decrease or even impair the invasive
capacity of the tumor cell as well as its ability for growth factor
shedding, resulting in reduced invasiveness and metastatic
potential of the tumor. Thus, targeting CERT might offer a novel
way of preventing late-stage tumorgenesis, especially the
conversion from a benign/solid nodule to an aggressive,
metastasizing tumor.
[0046] For therapeutic applications it is, thus, the goal to reduce
and/or inhibit the activity and/or expression of CERT. This can be
achieved either by a nucleotide composition which is used as human
therapeutic to treat a disease by inhibiting CERT function whereby
the drug is composed of an RNAi, and siRNA or an antisense RNA
specifically inhibiting CERT through binding a sequence motive of
CERT RNA. Reduction/inhibition of CERT activity/expression can also
be achieved by a drug substance containing nucleotides binding and
silencing the promoter of the CERT gene.
[0047] Furthermore, a drug substance or product can be composed of
a new chemical entity or peptide or protein inhibiting CERT
expression or activity. In case of a protein being the active
pharmaceutical compound it may be a (i) protein binding to CERT
promoter thereby inhibiting CERT expression, (ii) protein binding
to CERT or PKD thus preventing binding of PKD and CERT and
hindering CERT phosphorylation by PKD, (iii) a protein similar to
CERT which however does not fulfill CERT functions, that means a
"dominant-negative" CERT variant, or (iv) a protein acting as
scaffold for both CERT and PKD, resulting in irreversible binding
of is CERT to PKD (=a stable PKD/CERT complex) which is not
functional due to the inhibitory phosphorylation of CERT by PKD and
the hindering of dissociation of CERT from said complex.
SUMMARY OF THE INVENTION
[0048] The present invention is not obvious from the prior art. Up
to this point the only experimental data available on the protein
CERT pointed to a role in transport of ceramide from the
endoplasmic reticulum to the Golgi apparatus as a precursor of
sphingomyelin. Only the data described in this invention lead to a
novel working model for a role of CERT in protein transport form
the Golgi to the plasma membrane in eukaryotic cells. The prior art
does not give any hint on the possibility of enhancing the rate of
secretory transport of proteins in eukaryotic cell lines by
introducing the gene encoding CERT or another member of the START
domain protein family.
[0049] The surprising and unexpected working model of the present
invention identifies CERT as a novel in vivo PKD substrate and
crucial regulator of Golgi function.
[0050] PKD is known from the prior art. It is a family of
serine/threonine-specific protein kinases comprising three
structurally related members: PKD1/PKC.mu., PKD2 and
PKD3/PKC.upsilon.. PKD contains two aminoterminal zinc finger-like
cysteine-rich motifs that bind DAG, a pleckstrin homology (PH)
domain that negatively regulates PKD enzymatic function and a
carboxyterminal kinase domain.
[0051] The three PKD isoforms localize to the cytosol, nucleus,
Golgi complex and plasma membrane, where they regulate diverse
cellular processes, ranging from proliferation, differentiation,
apoptosis, cytoskeletal reorganization and metastasis to vesicle
trafficking (reviewed in (Rykx et al., 2003; Wang, 2006)). Thus
far, only a few physiological PKD substrates are known, which
include the neuronal protein Kidins220, the Ras effector RIN1,
histone deacetylase 5, E-cadherin and PI4KIII.beta. (Iglesias et
al., 2000; Jaggi et al., 2005; Vega et al., 2004; Wang et al.,
2002). At the TGN, PKD is critically involved in the fission of
transport carriers en route to the cell surface (Liljedahl et al.,
2001; Yeaman et al., 2004). PKD is recruited to the TGN by its
cysteine-rich regions (Baron and Malhotra, 2002; Hausser et al.,
2002; Maeda et al., 2001), where it is activated by PKCc-mediated
phosphorylation (az Anel and Malhotra, 2005).
[0052] Recently PI4KIIIa was identified, a key player in structure
and function of the Golgi apparatus, as a PKD substrate at this
organelle (Hausser et al., 2005). PKD-mediated phosphorylation of
PI4KIIIa at serine 294 stimulates its lipid kinase activity,
resulting in enhanced phosphatidylinositol 4-phosphate (PI(4)P)
production and vesicular stomatitis virus G-protein transport to
the plasma membrane (Hausser et al., 2005).
[0053] Protein kinase D (PKD) has been identified as a crucial
regulator of secretory transport at the trans-Golgi-network (TGN).
Recruitment and activation of PKD at the TGN is mediated by the
lipid diacylglycerol (DAG), a pool of which is generated by
sphingomyelin synthase from ceramide and phosphatidylcholine. The
non-vesicular transfer of ceramide from the endoplasmic reticulum
to the Golgi complex is mediated by the lipid transfer protein
CERT. This is described for example in Hanada et al, 2003, Nature
Vol 426, 803-809 and Hanada 2006, Molecular and Cellular
Biochemistry 286, 23-31 as well as in the corresponding patent
applications WO2005004898 and EP1652530. In neither one of these
documents, however, Hanada shows or points towards an implication
of modulating CERT expression or activity (let alone other START
domain proteins) in a method of producing proteins for diagnostic,
research or therapeutic purposes. Furthermore, these
documents/patent applications do not describe in any is way the use
of a blocking agent which reduces or completely blocks CERT
expression or activity in a pharmaceutical composition. Hanada
rather concludes to use CERT itself as a drug to promote ceramide
transport.
[0054] The present invention, however, identifies CERT as a novel
in vivo PKD substrate. Phosphorylation on serine 132 by PKD
decreases the affinity of CERT towards its lipid target
phosphatidylinositol 4-phosphate at Golgi membranes and reduces
ceramide transfer activity, identifying PKD as a regulator of lipid
homeostasis. The present invention also shows that CERT in turn is
critical for PKD activation and PKD dependent protein cargo
transport to the plasma membrane. The interdependence of PKD and
CERT is thus a key to the maintenance of Golgi membrane integrity
and secretory transport.
DESCRIPTION OF THE FIGURES
[0055] FIG. 1: Intracellular Product Accumulation.
[0056] Increase of intracellular product during Fed-batch
fermentations shown for three processes. Fed-batch fermentation was
performed using three different CHO producer cell clones expressing
human IgG antibodies: Process A (circles), B (diamonds) and M
(triangles), respectively). Every other day, cell samples were
taken, fixed and subjected to direct immunofluorescence to detect
the antibody light-chain. The amount of product was measured by
FACS and plotted relative to the amount at day 1.
[0057] FIG. 2: The Start Domain Protein Family
[0058] Phylogenetic assembly of (A) human START domain proteins,
(B) their domain organization (4 TM, four transmembrane; Pre,
mitochondrial presequence; Thio, acyl-CoA thioesterase), and (C)
their homologs in fly and worm. (taken from (Soccio and Breslow,
2003))
[0059] FIG. 3: CERT is a Crucial Regulator of Golgi Function and
Acts Downstream of XBP-1 in the Secretory Pathway.
[0060] (A) CERT and PKD are connected in a regulatory
feedback-loop. The scheme summarizes the current working hypothesis
where PKD is activated by DAG and phosphorylates CERT.
Phosphorylated CERT dissembles from PI(4)P and releases ceramide at
the site of its destination. Ceramide at the Golgi is converted to
sphingomyelin and DAG which in turn is necessary for PKD
activation. This circuit can be interrupted by mutation of the CERT
phosphorylation site (S132A).
[0061] (B) The schematic drawing shows the way of a secreted
protein from transcription and translation through the ER and Golgi
compartments to the plasma membrane where the protein is finally
released from the cell into the medium. The arrows represent recent
genetic engineering approaches aiming to enhance protein
production. Most efforts focused on transcription enhancing
technologies, few on translation engineering, and at present, only
three examples have been reported which target proteins involved in
post-translational processing within the ER (BiP, PDI and XBP-1).
CERT acts downstream of the ER in the secretory pathway and thus to
our knowledge represents the first target for engineering at later
stages of the secretion process
[0062] FIG. 4: CERT is Detected by a PKD Substrate Antibody.
[0063] (A) HEK293T cells were transfected with expression plasmids
encoding Flag-tagged CERTL and CERT. Cells were lysed 24 h post
transfection and CERT isoforms were immunoprecipitated with
anti-Flag antibody. Immunoprecipitated proteins were subjected to
SDS-PAGE, followed by immunoblotting with PKD substrate antibody
(pMOTIF; top panel) and, after stripping, with anti-Flag antibody
(bottom panel).
[0064] (B) HEK293T cells were transfected with Flag-CERT expression
plasmid, along with GFP-PKD1 K612W (PKD-KD) or empty vector. CERT
was analyzed by Western blotting as described in (A). Expression of
PKD-KD was verified by immunoblotting with a PKD-specific is
antibody (C20; bottom panel).
[0065] (C) COS 7 cells were cotransfected with Flag-CERT and
PKD1-GFP expression plasmids, fixed and stained with Flag-specific
antibody (red). The images shown are stacks of several confocal
sections. Scale bar, 20 .mu.m.
[0066] FIG. 5: PKD Phosphorylates CERT on Serine 132.
[0067] (A) Alignment of the peptide sequences used to raise the PKD
substrate antibody and two potential PKD motifs in CERT.
[0068] (B) HEK293T cells were transfected with expression plasmids
encoding Flag-tagged CERT wild type (WT), CERT-S132A, and
CERT-S272A. The cells were lysed and CERT proteins were
immunoprecipitated and analyzed by Western blotting as described in
FIG. 4.
[0069] (C) Recombinant GST-Flag-CERT wild type (WT) and S132A
fusion proteins were incubated in kinase buffer containing
[32P]-a-ATP in the absence (-) and presence (+) of purified PKD1
for 30 min. Proteins were separated by SDS-PAGE and transferred to
membrane. Incorporation of radioactive phosphate was analyzed using
a PhosphoImager (top), followed by immunoblotting with
Flag-specific antibody to verify equal loading of the CERT
proteins.
[0070] (D) Recombinant CERT proteins were subjected to an in vitro
kinase with purified PKD1 as described in (C) in the presence of
cold ATP. Immunoblotting was performed with the pMOTIF antibody
and, after stripping, with Flag-specific antibody to verify equal
loading of the CERT proteins. PKD1 and CERT proteins are marked
with arrows; the bands with asterisks are due to non-specific
binding.
[0071] FIG. 6: CERT Phosphorylation on Serine 132 Modulates PI(4)P
Binding and Ceramide Transfer Activity.
[0072] HEK293T cells were transfected with expression plasmids
encoding GFPtagged CERT wild type is (WT, SEQ ID NO.10, 12) and
CERT-S132A (SEQ ID NO.14). Cells were harvested by hypotonic lysis
24 h post transfection and the cytosol fraction was recovered after
centrifugation at 100.000.times.g.
[0073] Samples containing equal amounts of GFP fluorescence were
used for (A) Protein-lipid overlay assays. Cytosol from HEK293T
cells transiently expressing the CERT variants was incubated with
membranes spotted with a concentration gradient of the different
phosphoinositides and bound CERT proteins were detected via their
GFP tag.
[0074] (B) Donor liposomes containing TNPPE and pyrene-ceramide
were mixed with a 10-fold excess of unlabeled acceptor liposomes.
After 60 sec, cytosol from cells transiently expressing GFP-tagged
CERT wild type (WT), S132A, or GFP alone (con) was added and pyrene
fluorescence at 395 nm was recorded (excitation: 340 nm). Spectra
were normalized to maximum fluorescence in Triton X-100 and to
maximum GFP fluorescence.
[0075] FIG. 7: CERT Regulates PKD Activation and Secretory
Transport.
[0076] (A) Western Blot of whole cell lysates from HEK293T cells
transfected with either Flag-tagged CERT wild type (SEQ ID NO.10,
12) or the CERT mutant S132A (SEQ ID NO. 14). The blot was probed
with phosphospecific pS916 PKD antibody (top panel), a PKD-specific
antibody (middle panel) and a Flag-specific antibody (bottom
panel), respectively, to verify expression of the Flag-tagged CERT
constructs.
[0077] (B) Measurement of HRP-activity in the supernatants of
HEK293T cells cotransfected with Flag-ss-HRP and empty vector
(black bars), PKD1-GFP kinase dead (KD, white bars), Flag-CERT wild
type (WT, shaded bars) or Flag-CERT-S132A (dark grey). Relative
light units (RLU) were plotted at the indicated time points after
medium change. The values correspond to the mean of triplicate
samples, error bars=SEM.
[0078] (C) Confocal immunofluorescence of GFP-CERT (green) and the
cis/medial-Golgi marker GS28 (red) in COS 7 cells. The images shown
are stacks of several confocal sections. Scale bar, 20 .mu.m.
[0079] (D) Stacks of confocal images showing the co-localization of
GFP-CERT (green) and HRP-Flag (red) in COS 7 cells. Scale bar, 20
.mu.m and 5 .mu.m (enlargement).
[0080] FIG. 8: CERT Downregulation by RNA Interference Inhibits
Secretory Transport.
[0081] (A) Quantitative detection of HRP activity in the
supernatants of COS 7 cells treated with either mock-(white),
lacZ-(light grey=lacZ-specific siRNA SEQ ID No 9) or CERT-specific
siRNA oligonucleotides (dark grey=siCERT#1 SEQ ID No 7 and
black=siCERT#2 SEQ ID No. 8). The relative light units (RLU) of
triplicate experiments are shown, error bars=SEM.
[0082] (B) Western Blot of the cell lysates of (A) probed with an
anti-transferrin receptor antibody. Equal loading was confirmed by
using an anti-Tubulin-specific antibody.
[0083] FIG. 9: Consensus Terms for the Start Domain
[0084] The consensus is given in relation to the number of
proteins, which fit to this consensus sequence and not in relation
to the number of amino acids which fit. That means that for the 80%
consensus sequence 80% of the START domain proteins compared have
the given amino acid at a particular position, e.g. a hydrophobic
amino acid abbreviated with "h".
[0085] This consensus sequence was generated by using the WEB-based
program "SMART" (see also Ponting & Aravind, 1999, TIBS 24,
pages 130-132).
[0086] (A) 80% consensus sequence (SEQ ID NO 28) for START domain
proteins.
[0087] (B) The START domain consensus sequence has been derived
from an amino acid alignment of START domain proteins. The
alignment includes 50%, 65% and 80% consensus sequences.
[0088] See the following amino acid grouping for help on
abbreviation and the corresponding classes.
Class Key Residues
[0089] alcohol o S, T aliphatic 1 I, L, V
any . A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
Y
[0090] aromatic a F, H, W, Y charged c D, E, H, K, R hydrophobic h
A, C, F, G, H, I, K, L, M, R, T, V, W, Y negative - D, E polar p C,
D, E, H, K, N, Q, R, S, T positive + H, K, R small s A, C, D, G, N,
P, S, T, V tiny u A, G, S turnlike t A, C, D, E, G, H, K, N, Q, R,
S, T
[0091] FIG. 10: Introduction of CERT Increases Monoclonal Antibody
Production
[0092] Expression constructs for Mock, CERT-WT or the mutant
CERT-SA were stably introduced into a CHO production cell line
secreting a human monoclonal IgG-type antibody. The effect of the
transgenes on the specific IgG productivity in these stable clones
was than measured (A) in serial stock cultures and (B) under
fed-batch production conditions as in FIG. 11 with n=3-4 for each
genotype. Error bars indicate standard deviations. One
representative result out of three independent experiments is
shown.
[0093] FIG. 11: Heterologous CERT Increases HSA Secretion
[0094] (A) Increased titer and specific productivity in serial
cultures. CHO cells secreting human serum albumine (HSA) were
stably transfected with either an empty plasmid ("Mock") CERT wild
type (CERT-WT) or the CERT mutant S132A (CERT-SA). From the
resulting stable cell pools (n=3 per genotype), the titer of HSA
was determined during 3-5 serial passages. The specific
productivity for HSA (black bars) and the titer (grey bars) were
calculated for each genotype and plottet as mean values of the
three pools. Error bars represent standard deviations.
[0095] (B) and (C) The cells from (A) were grown in shake-flasks
for 7 days and feeded every 24 hours from day 3 on. Samples from
the cell culture fluid were taken at day 3, 5 and 7 and subjected
to titer measurement of the recombinant HSA product. Specific
productivities (B) and titer (C) were calculated and plottet over
the time of fermentation. The following cells were compared: Mock
(-.quadrature.-), CERT-WT (-.tangle-solidup.-) and CERT-SA cells (-
-); error bars represent the standard deviations from three stable
pools per genotype.
LEGEND TO SEQUENCE LISTING
[0096] SEQ ID NO 1: PCR primer for human DNA CERT-S132A
[0097] SEQ ID NO 2: PCR primer for human DNA CERT-S132Arev
[0098] SEQ ID NO 3: PCR primer for human DNA CERT-S272A
[0099] SEQ ID NO 4: PCR primer for human DNA CERT-S272Arev
[0100] SEQ ID NO 5: PCR primer for human DNA CERT-138truncation
[0101] SEQ ID NO 6: PCR primer for human DNA
CERT-138truncationrev
[0102] SEQ ID NO 7: siRNA/DNA siCERT-1
[0103] SEQ ID NO 8: siRNA/DNA siCERT-2
[0104] SEQ ID NO 9: siRNA/DNA siLacZ
[0105] SEQ ID NO 10: human: CERT cDNA
[0106] SEQ ID NO 11: human: CERT protein
[0107] SEQ ID NO 12: human: CERT L cDNA
[0108] SEQ ID NO 13: human: CERT L protein
[0109] SEQ ID NO 14: human: CERT S132A cDNA
[0110] SEQ ID NO 15: human: CERT S132A protein
[0111] SEQ ID NO 16: human: START Domain CERT cDNA
[0112] SEQ ID NO 17: human: START Domain CERT protein
[0113] SEQ ID NO 18: human: START Domain CERT L cDNA
[0114] SEQ ID NO 19: human: START Domain CERT L protein
[0115] SEQ ID NO 20: human: StarD4 cDNA
[0116] SEQ ID NO 21: human: StarD4 protein
[0117] SEQ ID NO 22: human: StarD5 cDNA
[0118] SEQ ID NO 23: human: StarD5 protein
[0119] SEQ ID NO 24: human: StarD6 cDNA
[0120] SEQ ID NO 25: human: StarD6 protein
[0121] SEQ ID NO 26: human: PCTP cDNA
[0122] SEQ ID NO 27: human: PCTP protein
[0123] SEQ ID NO 28: START domain consensus sequence (FIG. 9)
DETAILED DESCRIPTION OF THE INVENTION
[0124] Post-translational modification of proteins by
phosphorylation is a common mechanism to induce conformational
changes that modulate enzymatic activity, mediate protein-protein
interactions or regulate subcellular localization. PKD is a key
regulator at the Golgi complex with PI4KIII.beta. being the only
local substrate identified thus far. To test whether the Golgi
complex-localized CERT protein may serve as a substrate for PKD, we
made use of a phosphospecific substrate antibody, termed pMOTIF,
raised against consensus motifs phosphorylated by PKD (Doppler et
al., 2005). HEK293T cells were transfected with expression vectors
encoding Flag-tagged CERT and CERT.sub.L. The CERT iso forms were
immunoprecipitated with Flag-specific antibodies and analyzed by
Western blotting with the pMOTIF antibody is (FIG. 4A). A pMOTIF
signal corresponding to the molecular weight of CERT and, more
weakly, to that of CERT.sub.L was detected (FIG. 4A). The weaker
detection of the phosphorylated CERT.sub.L iso form may be related
to its known behaviour to form aggregates, which may impact
phosphosite accessibility to kinases (Raga et al., 2000). To
investigate whether recognition of CERT by the pMOTIF antibody was
dependent upon PKD, we expressed CERT together with a kinase dead
variant of PKD1 (K621W) in HEK293T cells. This mutant has been
shown to localize to the Golgi complex and suppressed PI4KIII.beta.
phosphorylation in a dominant negative fashion (Hausser et al.,
2005). Coexpression of inactive PKD abolished detection of CERT
with the pMOTIF antibody, suggesting that the pMOTIF signal was
indeed due to PKD-mediated CERT phosphorylation (FIG. 4B). Lipid
transfer proteins are thought to act at MCS, which are formed
between the ER and TGN (Levine and Loewen, 2006), where PKD is
localized. Immunofluorescence staining of Flag-tagged CERT in COS 7
cells coexpressed with GFP-tagged PKD1 verified that the two
proteins colocalize at the Golgi complex (FIG. 4C). RNA
interference experiments suggest that simultaneous knock-down of
PKD1 and PKD2 was required to reduce CERT phosphorylation,
indicating that these two isoforms were primarily responsible for
phosphorylating CERT, whereas PKD3 appeared to play a minor role
(data not shown). This is in accordance with previously reported
overlapping substrate specificities of PKD1 and PKD2. For example,
PKD1 and PKD2 were both shown to phosphorylate PI4KIII.beta.,
whereas PKD3 failed to do so (Hausser et al., 2005).
[0125] To identify pMOTIF recognition sites in CERT, we searched
for potential PKD consensus motifs characterized by a leucine,
isoleucine or valine residue in the -5 and arginine in the -3
position relative to a serine or threonine. Two serines at
positions 132 and 272, matching the PKD consensus motif and
conserved across species (FIG. 5A), were exchanged for alanines by
site-directed mutagenesis. These mutants were expressed in HEK293T
cells and tested for recognition by the pMOTIF antibody.
Interestingly, mutation of serine 132 to alanine abrogated is
detection of CERT with the pMOTIF antibody and caused an increase
in electrophoretic mobility, indicative of loss of phosphorylation,
while the S272A mutation did not affect the pMOTIF signal (FIG.
5B). This suggested that serine 132 is a PKD phosphorylation site
specifically recognized by the PKD substrate antibody. To confirm
that PKD was capable of directly phosphorylating this serine
residue in CERT, we performed in vitro kinase assays with purified
PKD1 and recombinant CERT GST-fusion proteins produced in E. coli
comprising the first 138 amino acids of the protein. When the
truncated wild type CERT fusion protein was incubated with PKD1 in
the presence of [.gamma.-.sup.32P]-ATP, incorporation of
radioactivity was detected (FIG. 5C). This was significantly
impaired in the case of the CERT-S132A fusion protein. In vitro PKD
phosphorylation of wild type but not CERT-S132A is further shown to
generate a recognition site for the pMOTIF antibody (FIG. 5D).
Taken together, these results prove that CERT is a genuine PKD
substrate in vitro and in vivo and identify serine 132 as a
specific PKD phosphorylation site in CERT.
[0126] Serine 132 is in very close proximity to the CERT PH domain
(amino acids 23-117), making it possible that phosphorylation on
this site affects PI(4)P binding by increasing the local negative
charge. We therefore quantified PI(4)P binding of wild type CERT
and the CERT-S132A mutant by performing protein-lipid overlay
assays. Here, cytosol from HEK293T cells transiently expressing the
CERT variants was incubated with membranes spotted with a
concentration gradient of the different phosphoinositides and bound
CERT proteins were detected via their GFP tag. As reported
previously, the full-length wild type protein demonstrated weak
binding to several phospholipid species, but displayed strong
interaction with PI(4)P (Hanada et al., 2003; Levine and Munro,
2002). CERT-S132A binding to PI(4)P was detectable at two- to
fourfold lower concentrations as compared to that of the wild type
protein, suggesting increased affinity of the CERT-S132A mutant to
this phospholipid (FIG. 6A). Together, these data imply that is
CERT, once bound to the Golgi complex, is phosphorylated by PKD.
This then decreases the affinity of CERT to PI(4)P and thereby
regulates the interaction of CERT with Golgi membranes.
[0127] The CERT protein has been shown to function as a lipid
transfer protein (Hanada et al., 2003). We thus investigated
whether CERT phosphorylation on serine 132 influenced its ability
to bind and transfer ceramide between membranes. To this end,
GFP-tagged versions of wild type CERT and CERT-S132A were
transiently expressed in HEK239T cells and the cytosol fraction was
analyzed for ceramide-specific lipid transfer activity using a
FRET-based assay (FIG. 6B). In this assay, small unilamellar
vesicles containing pyrene-labeled ceramide as a fluorescent donor
and quenching amounts of head group-labeled TNP-PE were employed
(Olayioye et al., 2005; Somerharju, 2002). When these donor
liposomes were mixed with an excess of unlabeled acceptor
liposomes, the increase in pyrene fluorescence was negligible,
indicating minimal spontaneous ceramide transfer to acceptor
membranes (data not shown). Upon addition of wild type
CERT-containing cytosol, a steady increase in fluorescence was
noted, which was not observed when control cytosol of
vector-transfected cells was used (FIG. 6B). Compared to the wild
type protein, CERT-S 132A displayed a higher rate of lipid
transfer, evident from a more rapid increase in pyrene fluorescence
(FIG. 6B). This suggests that CERT phosphorylation on serine 132
downregulates ceramide transfer activity by decreasing association
of the protein with membranes. Previous data have already shown
that PKD regulates the level of PI(4)P at the Golgi complex by
phosphorylation-mediated activation of PI4KIII.beta. (Hausser et
al., 2005). Interestingly, PI4KIII.beta. is critical for the
transport of ceramide between the ER and the Golgi complex (Toth et
al., 2006). Accordingly, together with the data presented here, a
dual role for PKD in maintaining lipid homeostasis of Golgi
membranes becomes apparent by controlling the on-rate (via PI(4)P
levels) and the off-rate (via direct phosphorylation) of CERT.
[0128] The transfer of ceramide from the ER to the TGN is essential
for SM synthesis at this compartment (Hanada et al., 2003).
Golgi-localized SM synthase 1 (SMS1) utilizes ceramide and PC to
generate SM and DAG (Perry and Ridgway, 2005), the latter being a
prerequisite for PKD recruitment and activation (Baron and
Malhotra, 2002). Compounds that block DAG production at the TGN
inhibit the binding of PKD to TGN membranes and interfere with
secretory transport (Baron and Malhotra, 2002). Therefore,
increased ceramide transfer from the ER to the TGN by
overexpression of CERT should result in an elevated local DAG pool
and may consequently stimulate PKD activity and secretory
transport. To test this hypothesis, we transiently expressed CERT
wild type and CERT-S132A in HEK293T cells and analyzed
autophosphorylation of endogenous PKD. Compared to the control,
expression of both CERT wild type and CERT-S132A increased PKD
activity, as revealed by analyses with a phosphospecific PKD
antibody (FIG. 7A). This shows that PKD activation is regulated by
CERT proteins, likely due to increased ceramide delivery and
enforced SM/DAG synthesis. A similar function has recently been
described for the lipid transfer protein Nir2 in the maintenance of
DAG levels at the Golgi apparatus via regulation of the CDP-choline
pathway (Litvak et al., 2005). RNAi-mediated knock-down of Nir2
decreased the levels of DAG and PKD at the Golgi complex and
blocked secretory transport. Interestingly, this effect could be
rescued by the addition of exogenous C.sub.6-ceramide (Litvak et
al., 2005), indicating a critical role for ceramide in DAG
synthesis and PKD recruitment to the Golgi complex.
[0129] To address the question of whether CERT-mediated PKD
activation indeed translated into enhanced secretory transport, we
made use of a plasmid encoding horseradish peroxidase fused to a
signal sequence (ss). The fusion protein ss-HRP can be used as a
reporter for constitutive protein secretion (Bard et al., 2006). In
control cells, secretion of ss-HRP could be detected within 1 hour
and increased over time (FIG. 7B). Coexpression of kinase dead
PKD1, which inhibits secretory transport of cargo protein (Hausser
et al., 2005; Liljedahl et al., 2001), almost entirely abrogated
the secretion of ss-HRP into the supernatant. This confirmed that
HRP was secreted in a PKD-dependent manner in our assay.
Coexpression of CERT wild type and CERT-S132A strongly augmented
the amount of secreted HRP (FIG. 7B). Interestingly, we could only
detect a slight increase in secretion with the CERT-S132A mutant
compared to the one observed with the CERT wild type protein. This
is in accordance with the comparable activation of PKD by CERT and
CERT-S132A (FIG. 7A), but was unexpected in the light of the
significantly enhanced in vitro lipid transfer activity of the CERT
mutant (FIG. 6B). However, increased levels of ceramide may not
necessarily translate into equivalent increases in DAG, because DAG
synthesis might be limited by the availability of PC and the
activity of SM synthase. Accumulation of ceramide is known to
affect Golgi membrane stability and induces vesicle fission
(Fukunaga et al., 2000; Weigert et al., 1999). We therefore
investigated whether overexpression of the CERT-S132A mutant
affected its localization and/or caused morphological changes of
the Golgi apparatus. CERT has been demonstrated to colocalize with
the cis/medial-Golgi marker GS28 (Hanada et al., 2003).
Immunofluorescence analysis of GFP-tagged CERT expressed in COST
cells showed that the protein localized to GS28-positive Golgi
regions (FIG. 7C). By contrast, in addition to the partial
colocalization with GS28 at the Golgi complex, the CERT-S132A
mutant protein displayed a dispersed, punctate staining. Of note,
some of these vesicular structures were found to contain the cargo
protein ss-HRP, providing evidence that these structures indeed
represent Golgi-derived transport carriers (FIG. 7D). This finding
is in accordance with the observed changes in Golgi membrane
structure due to local increases in ceramide levels (Fukunaga et
al., 2000; Weigert et al., 1999).
[0130] In conclusion, we have identified CERT as a PKD substrate
and provide evidence for a novel relationship between membrane
lipid biogenesis and protein secretion. We show that CERT plays an
important role in vesicular transport processes by providing
ceramide as a substrate for the synthesis of the PKD activator DAG
at Golgi membranes. We further demonstrate that the is system is
tightly regulated by a negative feedback loop: Active PKD
phosphorylates CERT at serine 132, thus decreasing the affinity of
CERT towards its lipid target PI(4)P to ensure continuous rounds of
lipid transfer from the ER to the Golgi compartment.
[0131] The data of the present invention clearly demonstrate that
overexpression of CERT enhances protein secretion. To investigate
whether also the opposite is true, meaning that reduced CERT
expression would result in diminished secretion, siRNA experiments
were performed. The activity of HRP was detected after 3 hours and
showed equal comparable levels in both control cells. In contrast,
a dramatic reduction of HRP activity was measured in cells that had
been treated with any of the CERT-specific siRNA oligonucleotides
(FIG. 8). This indicates that reduced CERT levels lead to reduced
HRP secretion from the cells and further underscores the important
role of CERT in the secretory transport.
[0132] Interestingly, not only protein secretion, but also the
abundance of the transmembrane protein transferrin receptor was
affected by the reduction of CERT (FIG. 8B). When the cells from
FIG. 8A were pooled and the lysates probed with transferrin
receptor-specific antibodies in Western blot experiments, a strong
decrease in the amount of transferrin receptor became apparent,
whereas similar transferrin receptor levels were detected in both
control cells. This finding suggests, that the lipid transfer
protein CERT is not only implicated in the transport of secreted
but also of membrane-standing cell-surface proteins. This might not
be surprising as both types of proteins are equally transported in
lipid vesicles from the ER via the Golgi to the plasma membrane and
thus use the same cellular export routes which--as we demonstrate
in the present invention for the first time--are influenced by
CERT.
[0133] The findings and the resulting new model for regulation of
secretory protein transport from the Golgi complex to the plasma
membrane described in the present invention can be applied to
biopharmaceutical protein manufacturing. Overexpression of CERT
increases biopharmaceutical protein production of diverse proteins
such as antibodies, cytokines, growth factors such as
erythropoietin or insulin, surface receptors such as epithelial
growth factor, and membrane-bound proteases.
[0134] Although the method described in this invention can be
generally applied, to all protein production processes, the degree
of success of this strategy as measured by the increase in the
amount of protein produced can certainly depend on the particular
nature of the protein of interest. CHO or other producer cells are
transfected with an expression construct encoding a START domain
protein such as CERT, StarD4 or StarD5 or a mutant or derivative
thereof.
[0135] Notably, the highest titers are detected in cells expressing
unphosphorylatable CERT mutant S132A. Heterologous expression of
CERT, and especially mutant CERT, in CHO cells can enhance protein
secretion, for example of a monoclonal antibody, on the transient
transfection level. This can be particularly useful for fast
production of smaller quantities of drug candidates or drug targets
necessary in pharmaceutical research and development.
[0136] In a further embodiment of this invention, a producer cell
line is transfected with the same DNA constructs as above and
subsequently subjected to selection to obtain stable cell pools.
For six cell culture passages subsequent to the selection
procedure, culture supernatant is collected to be analysed for the
content of protein of interest. In case of a monoclonal antibody,
the concentration of the protein product is determined by ELISA and
divided by the mean number of cells to calculate the specific
productivity. Again, the highest values are seen in the cell pools
harbouring the CERT mutant. In cells containing a START domain
construct expression of the protein of interest is significantly
enhanced compared to MOCK or untransfected cells. Very similar
results can be obtained if the stable transfectants are subjected
to batch or fed-batch fermentations. In each of these settings,
overexpression of START domain proteins leads to enhanced
expression of antibodies, single cell proteins and surface
receptors in transiently as well as stably transfected CHO cell
lines, indicating that START domain proteins such as CERT is or
StarD4 and StarD5 are able to enhance the specific production
capacity of the cells under fermentation conditions.
DEFINITIONS
[0137] The general embodiments "comprising" or "comprised"
encompass the more specific embodiment "consisting of".
Furthermore, singular and plural forms are not used in a limiting
way.
[0138] Terms used in the course of this present invention have the
following meaning
[0139] The term "START domain" stands for steroidogenic acute
regulatory protein (StAR) related lipid transfer (START) domain.
This domain of about 200-210 amino acids was identified initially
as lipid binding domain (Soccio and Breslow, 2003; Tsujishita and
Hurley, 2000). The length of the START domain may vary between 116
to 250 amino acids, or between 180 to 223 amino acids, or more
specifically between 219 to 223 amino acids depending on the START
domain family member. The most striking feature of the START domain
structure is a predominantly hydrophobic tunnel extending nearly
the entire length of the protein which is used to binding a single
molecule of large lipophilic compounds, like cholesterol. The
structural resolution of the START domain family member MLN64-START
revealed an .alpha./.beta. type structure consisting of
nine-stranded twisted antiparallel .beta.-sheets and four
.alpha.-helices (Tsujishita and Hurley, 2000). The domain found in
various eukaryotic proteins is referred to as `classical START
domain` (CSD) while a similar domain specific to plants is known as
Birch allergen START domain (BA-START).
[0140] The term "CERT" encompasses both splice forms of CERT: CERT
(SEQ ID NO.11) and CERT.sub.L (SEQ ID No.13). The term "CERT"
furthermore encompasses any other possible splice form of CERT
derived from the nucleotide sequence SEQ ID No. 12.
[0141] The term "CERT" further encompasses hCERT protein and its
recombinants, hCERT, hCERTA, PH protein, hCERT A MR protein, and
hCERTA STprotein, and further, PHhCERT protein, MRhCERT protein and
SThCERT protein (see also EP1652530, (Hanada, 2006), (Hanada et
al., 2003)).
[0142] The term "derivative" in general includes sequences suitable
for realizing the intended use of the present invention, which
means that the sequences mediate the increase in secretory
transport in a cell.
[0143] The term "derivative" as used in the present invention means
a polypeptide molecule or a nucleic acid molecule which is at least
70% identical in sequence with the original sequence or its
complementary sequence. Preferably, the polypeptide molecule or
nucleic acid molecule is at least 80% identical in sequence with
the original sequence or its complementary sequence. More
preferably, the polypeptide molecule or nucleic acid molecule is at
least 90% identical in sequence with the original sequence or its
complementary sequence. Most preferred is a polypeptide molecule or
a nucleic acid molecule which is at least 95% identical in sequence
with the original sequence or its complementary sequence and
displays the same or a similar effect on secretion as the original
sequence.
[0144] Sequence differences may be based on differences in
homologous sequences from different organisms. They might also be
based on targeted modification of sequences by substitution,
insertion or deletion of one or more nucleotides or amino acids,
preferably 1, 2, 3, 4, 5, 7, 8, 9 or 10. Deletion, insertion or
substitution mutants may be generated using site specific
mutagenesis and/or PCR-based mutagenesis techniques. Corresponding
methods are described by (Lottspeich and Zorbas, 1998) in Chapter
36.1 with additional references. The sequence identity of a
reference sequence (in the present invention being for example
START domain SEQ ID No.16, 17 or 18, 19) can be determined by using
for example standard "alignment" algorithms, e.g. "BLAST"
((Altschul et al., 1990); (Madden et al., 1996); (Zhang and Madden,
1997)). Sequences are aligned when they fit together in their
sequence and are identifiable with the help of standard "alignment"
algorithms.
[0145] Furthermore, in the present invention the term "derivative"
means a nucleic acid molecule (single or double strand) which
hybridizes to SEQ ID No.10, 12, 14, 16, 18, 20, 22, 24, 26) or with
fragments or derivates thereof or with sequences which are
complementary to SEQ ID No. 10, 12, 14, 16, 18, 20, 22, 24, 26.
Preferably the hybridization is performed under stringent
hybridization- and washing conditions (e.g. hybridisation at
65.degree. C. in a buffer containing 5.times.SSC; washing at
42.degree. C. using 0.2.times.SSC/0.1% SDS). Corresponding
techniques are described exemplary in (Ausubel et al., 2002).
[0146] The term "derivatives" further means protein deletion
mutants, phosphorylation mutants especially at a serine, threonine
or tyrosine position, the deletion of a PKD binding site or the
CERT Ser132A mutation.
[0147] The term "activity" describes and quantifies the biological
functions of the protein within the cell or in in vitro assays.
[0148] An example of how to measure "activity" is described in the
patent application EP1652530 (Hanada et al.), which detects
ceramide release promotion activity from membranes. The lipid
membrane containing ceramide has to be prepared so that it contains
12.5 nCi (225 pmol) per sample of [palmitoyl-1-I4C]
N-palmitoyl-D-ethyro-sphigosine (hereinafter, may be referred to as
I4C-ceramide) on the basis of a mixed lipid consisting of
phosphatidylcholine and phosphatidylethanolamine at the ratio of
4:1 derived from egg yolk. Its concentration of ceramide thus is
2.5 mg/mL. For one sample of the activity measurement this lipid
membrane is required at an amount of 20 pL. After the amount of
lipid required for activity measurement has been dispensed in an
Eppendorf tube, it has to be dried by spraying nitrogen gas. After
this, the buffer 1 [20 mM Hepes-NaOH buffer (pH7.4) to which 50 mM
NaCl and 1 mM EDTA have been added] has to be added to the dried
lipid membrane, so that the concentration becomes 2.5 mg/mL. A
gently is supersonic treatment has to be performed using bath type
supersonic generator [Model 221 0 manufactured by Branson, Co.,
Ltd.]. The supersonic treatment has to be performed at 25.degree.
C. for 3 minutes. The sample then has to be mixed (vortex) for 30
seconds and then the supersonic treatment is repeated for 3
minutes. The lipid membrane prepared in this way is used in a
ceramide release assay. The ceramide release reaction for the lipid
membrane and its detection is performed as follows: CERT protein or
a recombinant protein thereof (under the standard conditions, the
amount of protein corresponding to 450 picomoles, which is 2-fold
molar equivalent amount of ceramide contained in the donating
membrane was used) is mixed up to 30 pL using buffer 2 [50 mM
Hepes-NaOH buffer (pH7.4) to which 100 mM NaCl and 0.5 mM EDTA have
been added]. Here, the reaction is initiated by adding 20 pL of
lipid membrane containing ceramide. The final concentration of
phospholipids is 1 mg/mL. Ceramide is contained at a ratio of about
0.3% comparing to the total phospholipid amount. After the mixture
of these has been incubated at 37.degree. C. for 30 minutes, it is
centrifuged at 50,000.times.g for 30 minutes and the lipid membrane
is precipitated. In the case where CERT protein from E. coli is
used, most of the protein remains in the supernatant under these
centrifugation conditions. Therefore, when I4C-ceramide binds to
CERT protein, it is releases from the lipid membrane and
transferred to the supernatant fraction. The activity for promoting
ceramide release with CERT is calculated by measuring the
radioactive activity of 1% in the supernatant fraction using a
liquid scintillation counter.
[0149] A further possibility to measure "activity" is an in vitro
ceramide transfer assay using recombinant material or cell lysate
containing CERT. Hereby, the protein-mediated transfer of ceramide
between SUVs is measured as described previously (Olayioye et al.,
2005). The transfer assay mixture contained donor vesicles (2 nmol
lipid/ml) composed of porcine brain lipids (Avanti Polar Lipids),
pyrene-labeled C.sub.16-ceramide, and
2,4,6-trinitrophenyl-phosphatidylethanolamine (TNP-PE) (88.6:0.4:11
mol %), provided by P. Somerharju, and a 10-fold excess of acceptor
vesicles composed of porcine brain lipids. Fluorescence intensity
is is recorded at 395 nm (excitation, 345 nm; slit widths, 4 nm)
before and after the addition of 75 .mu.g cytosol from HEK293T
cells transiently expressing the GFP-tagged CERT wild type and
S132A proteins (see above). Fluorescence intensities are normalized
to (i) the maximum intensity obtained after the addition of Triton
X-100 (0.5% final concentration) and (ii) the maximum GFP
fluorescence, to account for different protein expression
levels.
[0150] Another possibility to measure "activity" is a
phosphorylation state analysis of CERT S132A e.g. by using an
anti-phospho specific antibody in a Western blot. Whole cell
extracts are obtained by solubilizing cells in NP40 extraction
buffer (NEB) [50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM
sodium orthovanadate, 10 mM sodium fluoride, and 20 mM
.beta.-glycerophosphate plus Complete protease inhibitors]. Lysates
are clarified by centrifugation at 16,000.times.g for 10 min. Whole
cell extracts or immunoprecipitated proteins are boiled in sample
buffer and subjected to SDS-PAGE. The proteins are blotted onto
polyvinylidine difluoride membranes (Roth). After blocking with
0.5% blocking reagent (Roche) in PBS containing 0.1% Tween 20,
filters are probed with a phospho-specific antibody such as
phosphospecific substrate antibody, termed pMOTIF, raised against
consensus motifs phosphorylated by PKD (Doppler et al., 2005).
Proteins are visualized with peroxidase-coupled secondary antibody
using the enhanced chemiluminescence detection system (Pierce).
[0151] Still another assay for measuring the "activity" is a
secretion assay e.g. for a model protein, an antibody or a protein
of interest. Cells are cotransfected with ss-HRP-Flag plasmid and
empty vector, pEGFP-N1-PKD1KD and a plasmid encoding CERT, a
variant of CERT of any START family protein at a ratio of 1:6.5,
respectively. 24 h post-transfection cells are washed with
serum-free media and HRP secretion is quantified after 0, 1, 3 and
6 h by incubation of clarified cell supernatant with ECL reagent.
Measurements are done with a luminometer (Lucy2, Anthos) at 450
nm.
[0152] Another way to measure the "activity" is by using a
fluorescent ceramide analog e.g. Bodipy-labeled C5-ceramide,
perform chase experiments in intact cells and measure the
accumulation of is protein in the Golgi complex.
[0153] Quantification of the distribution of BODIPY.RTM. FL
C5-ceramide between the Golgi and the ER: The transport of the
fluorescent ceramide was quantified post-aquisition using the
linescan function of the Metamorph software. A line was drawn
through the cells in the confocal pictures taken in different time
points and the fluorescent intensity was measured in the cytoplasm
and over the Golgi complex of the cells. The "uptake ratio" was
calculated from the fluorescent light intensity in the Golgi
divided by the intensity measured in the cytoplasm. The maximum
uptake ratio was measured in control cells after 25 min incubation
on 37.degree. C. and this value was taken as 100 percent. The
quantification was made from the data of three independent
experiments in which confocal pictures were taken in twelve
different time points and in each time points 7 cells were
analyzed.
[0154] The term "productivity" or "specific productivity" describes
the quantity of a specific protein which is produced by a defined
number of cells within a defined time. The specific productivity is
therefore a quantitative measure for the capacity of cells to
express/synthesize/produce a protein of interest. In the context of
industrial manufacturing, the specific productivity is usually
expressed as amount of protein in picogram produced per cell and
day (`pg/cell*day` or `pcd`). One method to determine the "specific
productivity" of a secreted protein is to quantitatively measure
the amount of protein of interest secreted into the culture medium
by enzyme linked immunosorbent assay (ELISA). For this purpose,
cells are seeded into fresh culture medium at defined densities.
After a defined time, e.g. after 24, 48 or 72 hours, a sample of
the cell culture fluid is taken and subjected to ELISA measurement
to determine the titer of the protein of interest. The specific
productivity can be determined by dividing the titer by the average
cell number and the time.
[0155] Another example how to measure the "specific productivity"
of cells is provided by the homogenous time resolved fluorescence
(HTRF.RTM.) assay.
[0156] "Producitvity" of cells for an intracellular,
membran-associated or transmembrane protein can also be detected
and quantified by Western Blotting. The cells are first washed and
subsequently lysed in a buffer containing either detergents such as
Triton-X, NP-40 or SDS or high salt concentrations. The proteins
within the cell lysate are than separated by size on SDS-PAGE,
transferred to a nylon membrane where the protein of interest is
subsequently detected and visualized by using specific
antibodies.
[0157] Another method to determine the "specific productivity" of a
cell is to immunologically detect the protein of interest by
fluorescently labeled antibodies raised against the protein of
interest and to quantify the fluorescence signal in a flow
cytometer. In case of an intracellular protein, the cells are first
fixed, e.g. in paraformaldehyde buffer, and than permeabilized to
allow penetration of the detection antibody into the cell. Cell
surface proteins can be quantified on the living cell without need
for prior fixation or permeabilization.
[0158] The "productivity" of a cell can furthermore by determined
indirectly by measuring the expression of a reporter protein such
as the green fluorescent protein (GFP) which is expressed either as
a fusion protein with the protein of interest or from the same mRNA
as the protein of interest as part of a bi-, tri-, or multiple
expression unit.
[0159] The term "enhancement/increase of productivity" comprises
methods to increase/enhance the specific productivity of cells. The
specific productivity is increased or enhanced, if the productivity
is higher in the cells under investigation compared to the
respective control cells and if this difference is statistically
significant. The cells under investigation can be heterogenous
populations or clonal cell lines of treated, transfected or
genetically modified cells; untreated, untransfected or un-modified
cells can serve as control cells.
[0160] The terms "inhibitor" or "suppressor" as used in the present
invention means any molecule that acts to inhibit or suppress the
expression or activity of a START domain protein like CERT. The
term includes small chemical compounds, nucleic acids such as
antisense DNA, antisense RNA or siRNA, single chain antibodies and
proteins that block CERT transcription and translation as well as
peptides or proteins that interfere with lipid binding of START
domain proteins such as CERT.
[0161] "Host cells" in the meaning of the present invention are
cells such as hamster cells, preferably BHK21, BHK TK.sup.-, CHO,
CHO-K1, CHO-DUKX, CHO-DUKX B1, and CHO-DG44 cells or the
derivatives/progenies of any of such cell line. Particularly
preferred are CHO-DG44, CHO-DUKX, CHO-K1 and BHK21, and even more
preferred CHO-DG44 and CHO-DUKX cells. In a further embodiment of
the present invention host cells also mean murine myeloma cells,
preferably NS0 and Sp2/0 cells or the derivatives/progenies of any
of such cell line. Examples of murine and hamster cells which can
be used in the meaning of this invention are also summarized in
Table 1. However, derivatives/progenies of those cells, other
mammalian cells, including but not limited to human, mice, rat,
monkey, and rodent cell lines, or eukaryotic cells, including but
not limited to yeast, insect and plant cells, can also be used in
the meaning of this invention, particularly for the production of
biopharmaceutical proteins.
TABLE-US-00001 TABLE 1 Eukaryotic production cell lines CELL LINE
ORDER NUMBER NS0 ECACC No. 85110503 Sp2/0-Ag14 ATCC CRL-1581 BHK21
ATCC CCL-10 BHK TK.sup.- ECACC No. 85011423 HaK ATCC CCL-15
2254-62.2 (BHK-21 derivative) ATCC CRL-8544 CHO ECACC No. 8505302
CHO wild type ECACC 00102307 CHO-K1 ATCC CCL-61 CHO-DUKX ATCC
CRL-9096 (=CHO duk.sup.-, CHO/dhfr) CHO-DUKX B11 ATCC CRL-9010
CHO-DG44 (Urlaub et al., 1983) CHO Pro-5 ATCC CRL-1781 V79 ATCC
CCC-93 B14AF28-G3 ATCC CCL-14 HEK 293 ATCC CRL-1573 COS-7 ATCC
CRL-1651 U266 ATCC TIB-196 HuNS1 ATCC CRL-8644 CHL ECACC No.
87111906
[0162] Host cells are most preferred, when being established,
adapted, and completely cultivated under serum free conditions, and
optionally in media which are free of any protein/peptide of animal
origin. Commercially available media such as Ham's F12 (Sigma,
Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified
Eagle's Medium (DMEM; Sigma), Minimal Essential Medium (MEM;
Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO
(Invitrogen, Carlsbad, Calif.), CHO-S-Invtirogen), serum-free CHO
Medium (Sigma), and protein-free CHO Medium (Sigma) are exemplary
appropriate nutrient solutions. Any of the media may be
supplemented as necessary with a variety of compounds examples of
which are hormones and/or other growth factors (such as insulin,
transferrin, epidermal growth factor, insulin like growth factor),
salts (such as sodium chloride, calcium, magnesium, phosphate),
buffers (such as HEPES), nucleosides (such as adenosine,
thymidine), glutamine, glucose or other equivalent energy sources,
antibiotics, trace elements. Any other necessary supplements may
also be included at appropriate concentrations that would be known
to those skilled in the art. In the present invention the use of
serum-free medium is preferred, but media supplemented with a
suitable amount of serum can also be used for the cultivation of
host cells. For the growth and selection of genetically modified
cells expressing the selectable gene a suitable selection agent is
added to the culture medium.
[0163] The term "protein" is used interchangeably with amino acid
residue sequences or polypeptide and refers to polymers of amino
acids of any length. These terms also include proteins that are
post-translationally modified through reactions that include, but
are not limited to, glycosylation, acetylation, phosphorylation or
protein processing. Modifications and changes, for example fusions
to other proteins, amino acid sequence substitutions, deletions or
insertions, can be made in the structure of a polypeptide while the
molecule maintains its biological functional activity. For example
certain amino acid sequence substitutions can be made in a
polypeptide or its underlying nucleic acid coding sequence and a
protein can be obtained with like properties.
[0164] The term "polypeptide" means a sequence with more than 10
amino acids and the term "peptide" means sequences up to 10 amino
acids length.
[0165] The present invention is suitable to generate host cells for
the production of biopharmaceutical polypeptides/proteins. The
invention is particularly suitable for the high-yield expression of
a large number of different genes of interest by cells showing an
enhanced cell productivity.
[0166] "Gene of interest" (GOI), "selected sequence", or "product
gene" have the same meaning herein and refer to a polynucleotide
sequence of any length that encodes a product of interest or
"protein of interest", also mentioned by the term "desired
product". The selected sequence can is be full length or a
truncated gene, a fusion or tagged gene, and can be a cDNA, a
genomic DNA, or a DNA fragment, preferably, a cDNA. It can be the
native sequence, i.e. naturally occurring form(s), or can be
mutated or otherwise modified as desired. These modifications
include codon optimizations to optimize codon usage in the selected
host cell, humanization or tagging. The selected sequence can
encode a secreted, cytoplasmic, nuclear, membrane bound or cell
surface polypeptide.
[0167] The "protein of interest" includes proteins, polypeptides,
fragments thereof, peptides, all of which can be expressed in the
selected host cell. Desired proteins can be for example antibodies,
enzymes, cytokines, lymphokines, adhesion molecules, receptors and
derivatives or fragments thereof, and any other polypeptides that
can serve as agonists or antagonists and/or have therapeutic or
diagnostic use. Examples for a desired protein/polypeptide are also
given below.
[0168] In the case of more complex molecules such as monoclonal
antibodies the GOI encodes one or both of the two antibody
chains.
[0169] The "product of interest" may also be an antisense RNA.
[0170] "Proteins of interest" or "desired proteins" are those
mentioned above. Especially, desired proteins/polypeptides or
proteins of interest are for example, but not limited to insulin,
insulin-like growth factor, hGH, tPA, cytokines, such as
interleukines (IL), e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or
IFN tau, tumor necrosisfactor (TNF), such as TNF alpha and TNF
beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF. Also
included is the production of erythropoietin or any other hormone
growth factors. The method according to the invention can also be
advantageously used for production of antibodies or fragments
thereof. Such fragments include e.g. Fab fragments is (Fragment
antigen-binding=Fab). Fab fragments consist of the variable regions
of both chains which are held together by the adjacent constant
region. These may be formed by protease digestion, e.g. with
papain, from conventional antibodies, but similar Fab fragments may
also be produced in the mean time by genetic engineering. Further
antibody fragments include F(ab')2 fragments, which may be prepared
by proteolytic cleaving with pepsin.
[0171] The protein of interest is preferably recovered from the
culture medium as a secreted polypeptide, or it can be recovered
from host cell lysates if expressed without a secretory signal. It
is necessary to purify the protein of interest from other
recombinant proteins and host cell proteins in a way that
substantially homogenous preparations of the protein of interest
are obtained. As a first step, cells and/or particulate cell debris
are removed from the culture medium or lysate. The product of
interest thereafter is purified from contaminant soluble proteins,
polypeptides and nucleic acids, for example, by fractionation on
immunoaffinity or ion-exchange columns, ethanol precipitation,
reverse phase HPLC, Sephadex chromatography, chromatography on
silica or on a cation exchange resin such as DEAE. In general,
methods teaching a skilled person how to purify a protein
heterologous expressed by host cells, are well known in the art.
Such methods are for example described by (Harris and Angal, 1995)
or (Robert Scopes, 1988).
[0172] Using genetic engineering methods it is possible to produce
shortened antibody fragments which consist only of the variable
regions of the heavy (VH) and of the light chain (VL). These are
referred to as Fv fragments (Fragment variable=fragment of the
variable part). Since these Fv-fragments lack the covalent bonding
of the two chains by the cysteines of the constant chains, the Fv
fragments are often stabilised. It is advantageous to link the
variable regions of the heavy and of the light chain by a short
peptide fragment, e.g. of 10 to 30 amino acids, preferably 15 amino
acids. In this way a single peptide strand is obtained consisting
of VH and VL, linked by a is peptide linker. An antibody protein of
this kind is known as a single-chain-Fv (scFv). Examples of
scFv-antibody proteins of this kind known from the prior art are
described in (Huston et al., 1988).
[0173] In recent years, various strategies have been developed for
preparing scFv as a multimeric derivative. This is intended to
lead, in particular, to recombinant antibodies with improved
pharmacokinetic and biodistribution properties as well as with
increased binding avidity. In order to achieve multimerisation of
the scFv, scFv were prepared as fusion proteins with
multimerisation domains. The multimerisation domains may be, e.g.
the CH3 region of an IgG or coiled coil structure (helix
structures) such as Leucin-zipper domains. However, there are also
strategies in which the interaction between the VH/VL regions of
the scFv are used for the multimerisation (e.g. dia-, tri- and
pentabodies). By diabody the skilled person means a bivalent
homodimeric scFv derivative. The shortening of the Linker in an
scFv molecule to 5-10 amino acids leads to the formation of
homodimers in which an inter-chain VH/VL-superimposition takes
place. Diabodies may additionally be stabilised by the
incorporation of disulphide bridges. Examples of diabody-antibody
proteins from the prior art can be found in (Perisic et al.,
1994).
[0174] By minibody the skilled person means a bivalent, homodimeric
scFv derivative. It consists of a fusion protein which contains the
CH3 region of an immunoglobulin, preferably IgG, most preferably
IgG1 as the dimerisation region which is connected to the scFv via
a Hinge region (e.g. also from IgG1) and a Linker region. Examples
of minibody-antibody proteins from the prior art can be found in
(Hu et al., 1996).
[0175] By triabody the skilled person means a: trivalent
homotrimeric scFv derivative (Kortt et al., 1997). ScFv derivatives
wherein VH-VL are fused directly without a linker sequence lead to
the formation of trimers.
[0176] By "scaffold proteins" a skilled person means any functional
domain of a protein that is coupled by genetic cloning or by
co-translational processes with another protein or part of a
protein that has another function.
[0177] The skilled person will also be familiar with so-called
miniantibodies which have a bi-, tri- or tetravalent structure and
are derived from scFv. The multimerisation is carried out by di-,
tri- or tetrameric coiled coil structures (Lovejoy et al., 1993;
Pack et al., 1993; Pack et al., 1995).
[0178] By definition any sequences or genes introduced into a host
cell are called "heterologous sequences" or "heterologous genes" or
"transgenes" with respect to the host cell, even if the introduced
sequence or gene is identical to an endogenous sequence or gene in
the host cell.
[0179] A "heterologous" protein is thus a protein expressed from a
heterologous sequence.
[0180] Heterologous gene sequences can be introduced into a target
cell by using an "expression vector", preferably an eukaryotic, and
even more preferably a mammalian expression vector. Methods used to
construct vectors are well known to a person skilled in the art and
described in various publications. In particular techniques for
constructing suitable vectors, including a description of the
functional components such as promoters, enhancers, termination and
polyadenylation signals, selection markers, origins of replication,
and splicing signals, are reviewed in considerable details in
(Sambrook et al., 1989) and references cited therein. Vectors may
include but are not limited to plasmid vectors, phagemids, cosmids,
artificial/mini-chromosomes (e.g. ACE), or viral vectors such as
baculovirus, retrovirus, adenovirus, adeno-associated virus, herpes
simplex virus, retroviruses, bacteriophages. The eukaryotic
expression vectors will typically contain also prokaryotic
sequences that facilitate the propagation of the vector in bacteria
such as an origin of replication and antibiotic resistance genes
for selection in is bacteria. A variety of eukaryotic expression
vectors, containing a cloning site into which a polynucleotide can
be operatively linked, are well known in the art and some are
commercially available from companies such as Stratagene, La Jolla,
Calif.; Invitrogen, Carlsbad, Calif.; Promega, Madison, Wis. or BD
Biosciences Clontech, Palo Alto, Calif.
[0181] In a preferred embodiment the expression vector comprises at
least one nucleic acid sequence which is a regulatory sequence
necessary for transcription and translation of nucleotide sequences
that encode for a peptide/polypeptide/protein of interest.
[0182] The term "expression" as used herein refers to transcription
and/or translation of a heterologous nucleic acid sequence within a
host cell. The level of expression of a desired product/protein of
interest in a host cell may be determined on the basis of either
the amount of corresponding mRNA that is present in the cell, or
the amount of the desired polypeptide/protein of interest encoded
by the selected sequence as in the present examples. For example,
mRNA transcribed from a selected sequence can be quantitated by
Northern blot hybridization, ribonuclease RNA protection, in situ
hybridization to cellular RNA or by PCR (see (Sambrook et al.,
1989); (Ausubel et al., 2002) updated). Proteins encoded by a
selected sequence can be quantitated by various methods, e.g. by
ELISA, by Western blotting, by radioimmunoassays, by
immunoprecipitation, by assaying for the biological activity of the
protein, by immunostaining of the protein followed by FACS analysis
(see (Sambrook et al., 1989); (Ausubel et al., 2002) updated) or by
homogeneous time-resolved fluorescence (HTRF) assays.
[0183] "Transfection" of eukaryotic host cells with a
polynucleotide or expression vector, resulting in genetically
modified cells or transgenic cells, can be performed by any method
well known in the art and described, e.g., in (Sambrook et al.,
1989) or (Ausubel et al., 2002) updated. Transfection methods
include but are not limited to liposome-mediated transfection,
calcium is phosphate co-precipitation, electroporation, polycation
(such as DEAE-dextran)-mediated transfection, protoplast fusion,
viral infections and microinjection. Preferably, the transfection
is a stable transfection. The transfection method that provides
optimal transfection frequency and expression of the heterologous
genes in the particular host cell line and type is favoured.
Suitable methods can be determined by routine procedures. For
stable transfectants the constructs are either integrated into the
host cell's genome or an artificial chromosome/mini-chromosome or
located episomally so as to be stably maintained within the host
cell.
[0184] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology,
molecular biology, cell culture, immunology and the like which are
in the skill of one in the art. These techniques are fully
disclosed in the current literature. See e.g. Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel
et al., Current Protocols in Molecular Biology (1987, updated);
Brown ed., Essential Molecular Biology, IRL Press (1991); Goeddel
ed., Gene Expression Technology, Academic Press (1991); Bothwell et
al. eds., Methods for Cloning and Analysis of Eukaryotic Genes,
Bartlett Publ. (1990); Wu et al., eds., Recombinant DNA
Methodology, Academic Press (1989); Kriegler, Gene Transfer and
Expression, Stockton Press (1990); McPherson et al., PCR: A
Practical Approach, IRL Press at Oxford University Press (1991);
Gait ed., Oligonucleotide Synthesis (1984); Miller & Calos
eds., Gene Transfer Vectors for Mammalian Cells (1987); Butler ed.,
Mammalian Cell Biotechnology (1991); Pollard et al., eds., Animal
Cell Culture, Humana Press (1990); Freshney et al., eds., Culture
of Animal Cells, Alan R. Liss (1987); Studzinski, ed., Cell Growth
and Apoptosis, A Practical Approach, IRL Press at Oxford University
Presss (1995); Melamed et al., eds., Flow Cytometry and Sorting,
Wiley-Liss (1990); Current Protocols in Cytometry, John Wiley &
Sons, Inc. (updated); Wirth & Hauser, Genetic Engineering of
Animals Cells, in: Biotechnology Vol. 2, Puhler ed., VCH, Weinheim
663-744; the series Methods of Enzymology (Academic Press, Inc.),
and Harlow et al., eds., Antibodies: A Laboratory Manual
(1987).
EMBODIMENTS
[0185] The invention relates to a method of producing a
heterologous protein of interest in a cell comprising increasing
the expression or activity of a protein having an amino acid
sequence comprising a steroidogenic acute regulatory related lipid
transfer (START) domain or a derivative or mutant thereof, and
effecting the expression of said protein of interest. In a
preferred embodiment of the present invention the method is
characterized in that the heterologous protein is a membrane or
secreted protein.
[0186] In a specific embodiment of the present invention the method
is characterized in that the START domain protein is a mammalian
START domain family member such as PCTP (SEQ ID NO. 27), StarD7,
GPBP, StarD10, StarD8, StarD13, DLC-1, StarD4 (SEQ ID NO. 21),
StarD6 (SEQ ID NO. 25), StarD5 (SEQ ID NO. 23), MLN64, StAR,
THEA-2, CACH or StarD9 or a derivative or mutant thereof.
[0187] In a further specific embodiment of the present invention
the method is characterized in that the START domain protein is
characterized by being induced upon ER stress and/or is
structurally characterized by consisting solely of a START domain
such as StarD4 (SEQ ID NO. 21), StarD5 (SEQ ID NO. 23), StarD6 (SEQ
ID NO. 25) or phosphatidylcholin transfer protein (PCTP) (SEQ ID
NO. 27).
[0188] In another specific embodiment of the present invention the
method is characterized in that the START domain protein is
selected from the group consisting of CERT (SEQ ID NO. 11 or 13),
StarD4 (SEQ ID NO. 21) and StarD5 (SEQ ID NO. 23).
[0189] In a further embodiment of the present invention the method
is characterized in that the START is domain protein is StarD6 (SEQ
ID NO. 25). In a preferred embodiment StarD6 is encoded by a
nucleotide with the SEQ ID NO. 24.
[0190] In a preferred embodiment of the present invention the
method is characterized in that the START domain comprises at least
the 219 amino acid START domain of CERT.sub.L (SEQ ID NO. 19), or
at least the 223 amino acid START domain of CERT and CERT S132A
(SEQ ID NO. 17), or at least the START domain of StarD4 (SEQ ID NO.
21) or at least the START domain of StarD5 (SEQ ID NO. 23) or a
derivative or mutant thereof.
[0191] In a particularly preferred embodiment of the present
invention the method is characterized in that the START domain
protein is ceramide transfer protein CERT (CERT=SEQ ID NO. 11 or
CERT.sub.L=SEQ ID NO. 13) or a derivative or mutant thereof.
[0192] In another specific embodiment of the present invention the
method is characterized in that the START domain protein is mutated
ceramide transfer protein CERT and said mutation disables and/or
deletes a phosphorylation site at any serine, threonine or tyrosine
position of CERT.
[0193] In a further specific embodiment of the present invention
the method is characterized in that the START domain protein is
mutated ceramide transfer protein CERT and said mutation disables
and/or deletes the protein kinase D (PKD) phosphorylation site of
CERT at position 132.
[0194] In a particularly preferred embodiment of the present
invention the method is characterized in that the mutated CERT is
CERT.sub.S132A (SEQ ID NO. 15).
[0195] In another embodiment of the present invention the method is
characterized in that said method results in increased specific
cellular productivity of said protein of interest in said cell in
comparison to a control cell expressing said protein of interest,
but whereby said control cell does not have increased expression or
activity of a protein having an amino acid sequence comprising a
steroidogenic acute regulatory related lipid transfer (START)
domain or a derivative or mutant thereof.
[0196] In another specific embodiment of the present invention the
method is characterized in that the increase in productivity is
about 5% to about 10%, about 11% to about 20%, about 21% to about
30%, about 31% to about 40%, about 41% to about 50%, about 51% to
about 60%, about 61% to about 70%, about 71% to about 80%, about
81% to about 90%, about 91% to about 100%, about 101% to about
149%, about 150% to about 199%, about 200% to about 299%, about
300% to about 499%, or about 500% to about 1000%.
[0197] In a preferred embodiment of the present invention the
method is characterized in that said cell is a eukaryotic cell such
as a yeast, plant, worm, insect, avian, fish, reptile or mammalian
cell. In a specific embodiment of the present invention the method
is characterized in that said cell is an animal cell. In a further
specific embodiment of the present invention the method is
characterized in that said cell is a metazoan cell such as C.
elegans. In another specific embodiment of the present invention
the method is characterized in that said cell is a bilateria cell
such as Drosophila melanogaster. In a further embodiment of the
present invention the method is characterized in that said cell is
a vertebrate cell such as an avian, fish, reptile or mammalian
cell. In a specific embodiment of the present invention the method
is characterized in that said cell is a human cell such as the
human myeloma celline U266, HEK293, HeLa, HepG2 or HT1080. In a
preferred embodiment of the present invention the method is
characterized in that said cell is a rodent cell such as murine
NSO, Sp2/0 or Ag8653 cell, YO or YB2.0.
[0198] In a further embodiment of the present invention the method
is characterized in that said eukaryotic cell is a mammalian
cell.
[0199] In a specific embodiment of the present invention the method
is characterized in that said mammalian cell is a Chinese Hamster
Ovary (CHO), monkey kidney CV1, monkey kidney COS, human lens
epitheliaim PER.C6.TM., human embryonic kidney, HEK293, baby
hamster kidney, African green monkey kidney, human cervical
carcinoma, canine kidney, buffalo rat liver, human lung, human
liver, mouse mammary tumor or myeloma cell, a dog, pig or macaque
cell, rat, rabbit, cat, goat, preferably a CHO cell.
[0200] In a preferred embodiment of the present invention the
method is characterized in that said CHO cell is CHO wild type, CHO
K1, CHO DG44, CHO DUKX-B11, CHO Pro-5, preferably CHO DG44.
[0201] In a specific embodiment of the present invention the method
is characterized in that the protein of interest is a membrane or
secreted protein. In a preferred embodiment of the present
invention the method is characterized in that the protein of
interest is an antibody or antibody fragment.
[0202] In a further preferred embodiment of the present invention
the method is characterized in that the antibody is monoclonal,
polyclonal, mammalian, murine, chimeric, humanized, primatized,
primate, human or an antibody fragment or derivative thereof such
as antibody, immunoglobulin light chain, immunoglobulin heavy
chain, immunoglobulin light and heavy chains, Fab, F(ab')2, Fc,
Fc-Fc fusion proteins, Fv, single chain Fv, single domain Fv,
tetravalent single chain Fv, disulfide-linked Fv, domain deleted,
minibody, diabody, or a fusion polypeptide of one of the is above
fragments with another peptide or polypeptide, Fc-peptide fusion,
Fc-toxine fusion, scaffold proteins.
[0203] The invention further relates to a method for increasing
secretion of a membrane or secreted protein of interest in a cell
comprising expressing said protein of interest and expressing a
protein having an amino acid sequence comprising a steroidogenic
acute regulatory related lipid transfer (START) domain or a
derivative or mutant thereof.
[0204] The invention further relates to a method of producing a
membrane or secreted protein of interest in a cell comprising
increasing the expression of a protein having an amino acid
sequence comprising a steroidogenic acute regulatory related lipid
transfer (START) domain or a derivative or mutant thereof, and
effecting the expression of said protein of interest, whereby the
order or steps a and b may be reversed.
[0205] In a specific embodiment of the present invention the method
is further characterized in that step a) is carried out before step
b). In a further specific embodiment of the present invention the
method is further characterized in that step a) and b) are carried
out simultaneously. In another embodiment of the present invention
the method is further characterized in that step b) is carried out
before step a).
[0206] In a preferred embodiment of the present invention the
method further comprises an additional step of recovering the
protein of interest.
[0207] In an especially preferred embodiment of the present
invention the method further comprises an additional step of
isolating and purifying the protein of interest.
[0208] In a specific embodiment of the present invention the method
comprises increasing the is expression of a protein having an amino
acid sequence comprising a steroidogenic acute regulatory related
lipid transfer (START) domain or a derivative or mutant thereof by
transfecting a cell with a polynucleotide encoding for a protein
having an amino acid sequence comprising a steroidogenic acute
regulatory related lipid transfer (START) domain or a derivative or
mutant thereof.
[0209] In a specific embodiment of the present invention the method
comprises transfecting said cell with a first polynucleotide
encoding for a protein having an amino acid sequence comprising a
steroidogenic acute regulatory related lipid transfer (START)
domain or a derivative or mutant thereof and transfecting said cell
with a second polynucleotide encoding for a protein of
interest.
[0210] In a specific embodiment of the present invention the START
domain protein of the method is characterized by being induced upon
ER stress and/or is structurally characterized by having no further
structural motifs besides the START domain such as StarD4 (SEQ ID
NO. 21), StarD5 (SEQ ID NO: 23), StarD6 (SEQ ID NO. 25) or PCTP
(SEQ ID NO: 27).
[0211] In a preferred embodiment of the present invention the
method comprises increasing the expression of a protein having an
amino acid sequence comprising a steroidogenic acute regulatory
related lipid transfer (START) domain or a derivative or mutant
thereof, preferably by transfecting said cell with a first
polynucleotide encoding for a protein having an amino acid sequence
comprising a steroidogenic acute regulatory related lipid transfer
(START) domain or a derivative or mutant thereof, whereby the
increase is measured in comparison to an untransfected cell,
transfecting said cell with a second polynucleotide encoding for a
protein of interest
[0212] In a preferred embodiment of the present invention the
method is characterized by that the is proteins expressed in step
a) and b) are not identical.
[0213] The invention further relates to a method of producing a
membrane or secreted protein of interest in a cell comprising
[0214] Increasing the expression of a protein having an amino acid
sequence comprising a steroidogenic acute regulatory related lipid
transfer (START) domain or a derivative or mutant thereof in said
cell and effecting the expression of said protein of interest in
said cell.
[0215] The invention furthermore relates to a method of producing a
membrane or secreted protein of interest in a cell comprising
increasing the expression of a protein having an amino acid
sequence comprising a steroidogenic acute regulatory related lipid
transfer (START) domain or a derivative or mutant thereof in said
cell and expressing said protein of interest in said cell.
[0216] In a specific embodiment of the present invention the method
is characterized in that said method results in increased specific
cellular productivity of said protein of interest in said cell in
comparison to a control cell previously transfected with a
polynucleotide encoding for the protein of interest, but whereby
said control cell does not have increased expression of a protein
having an amino acid sequence comprising a steroidogenic acute
regulatory related lipid transfer (START) domain or a derivative or
mutant thereof.
[0217] In a specific embodiment of the present invention the method
is characterized in that the protein of interest is a protein which
is passing through the Golgi complex.
[0218] The invention further relates to a method of increasing
specific cellular productivity of a membrane or secreted protein of
interest in a cell comprising introducing into a cell one or more
vector systems comprising nucleic acid sequences encoding for at
least two polypeptides is whereby a first polynucleotide encodes a
protein having an amino acid sequence comprising a steroidogenic
acute regulatory related lipid transfer (START) domain or a
derivative or mutant thereof and a second polynucleotide encodes a
protein of interest and whereby the protein of interest and the
protein having an amino acid sequence comprising a steroidogenic
acute regulatory related lipid transfer (START) domain or a
derivative or mutant thereof are expressed by said cell.
[0219] The invention furthermore relates to a method of increasing
the transfection efficiency of a cell expressing a membrane or
secreted protein of interest in a cell comprising transfecting said
cell with a first polynucleotide encoding a protein having an amino
acid sequence comprising a steroidogenic acute regulatory related
lipid transfer (START) domain or a derivative or mutant thereof,
subsequently transfecting said cell with a second polynucleotide
encoding a protein of interest, whereby said first and second
polynucleotides are located on different vector systems.
[0220] In a further embodiment the invention relates to a method of
increasing the transfection efficiency of a cell comprising the
additional step of transfecting a reporter gene such as GFP, YFP,
HRP, SEAP or LacZ, which might be fused to the protein of interest,
located on the same expression construct or on a separate
plasmid.
[0221] In a preferred embodiment the invention relates to a method
of increasing the transfection efficiency of a cell comprising the
additional step of detecting and/or measuring the transfection
efficiency by either detection of the protein of interest or the
expression of the reporter gene.
[0222] The invention further relates to an expression vector
comprising two polynucleotides, a first polynucleotide encoding for
a protein having an amino acid sequence comprising a steroidogenic
is acute regulatory related lipid transfer (START) domain or a
derivative or mutant thereof and a second polynucleotide encoding
for a protein of interest.
[0223] In a specific embodiment of the present invention the
expression vector is characterized in that the START domain protein
is a mammalian START domain family member such as PCTP (SEQ ID NO.
27), StarD7, GPBP, StarD10, StarD8, StarD13, DLC-1, StarD4 (SEQ ID
NO. 21), StarD6 (SEQ ID NO. 25), StarD5 (SEQ ID NO. 23), MLN64,
StAR, THEA-2, CACH or StarD9 or a derivative or mutant thereof.
[0224] In another embodiment of the present invention the
expression vector is characterized in that the START domain protein
is ceramide transfer protein CERT (CERT=SEQ ID NO. 11 or
CERT.sub.L=SEQ ID NO. 13) or a derivative or mutant thereof.
[0225] In a specific embodiment of the present invention the
expression vector is characterized in that the mutated CERT is
CERT.sub.S132A (SEQ ID NO. 15).
[0226] In a specific embodiment of the present invention the
expression vector is characterized in that said first
polynucleotide increases the protein transport in a cell via the
secretory pathway.
[0227] In a specific embodiment of the present invention the
expression vector is characterized in that the START domain protein
is mutated ceramide transfer protein CERT and said mutation
disables and/or deletes a phosphorylation site at any serine,
threonine or tyrosine position within the CERT protein.
[0228] In another embodiment of the present invention the
expression vector is characterized in that the START domain protein
is mutated ceramide transfer protein CERT and said mutation
disables is and/or deletes the protein kinase D (PKD)
phosphorylation site of CERT at position 132.
[0229] The present invention further relates to a cell comprising
the expression vector of the invention. In a specific embodiment of
the present invention the cell is characterized in that said cell
is a eukaryotic cell such as a yeast, plant, worm, insect, avian,
fish, reptile or mammalian cell. In a specific embodiment of the
present invention the cell is characterized in that said eukaryotic
cell is a mammalian cell.
[0230] In a specific embodiment of the present invention the cell
is characterized in that said mammalian cell is a Chinese Hamster
Ovary (CHO), monkey kidney CV1, monkey kidney COS, human lens
epitheliaim PER.C6.TM., human embryonic kidney, HEK 293, baby
hamster kidney, African green monkey kidney, human cervical
carcinoma, canine kidney, buffalo rat liver, human lung, human
liver, mouse mammary tumor or myeloma cell, a dog, pig or macaque
cell, rat, rabbit, cat, goat, preferably a CHO cell. In a specific
embodiment of the present invention the cell is characterized in
that said CHO cell is CHO wild type, CHO K1, CHO DG44, CHO
DUKX-B11, CHO Pro-5, preferably CHO DG44.
[0231] In a specific embodiment of the present invention the cell
is characterized in that said cell is an animal cell, preferably a
metazoan cell such as C. elegans. In a further embodiment of the
present invention the cell is characterized in that said cell is a
bilateria cell such as Drosophila melanogaster, preferably a
vertebrate cell such as an avian, fish, reptile or mammalian cell.
In a specific embodiment of the present invention the cell is
characterized in that said eukaryotic cell is a mammalian cell,
preferably a human cell such as a the human myeloma celline U266,
HEK293, HeLa, HepG2 or HT1080, more preferably a rodent cell such
as murine NSO, Sp2/0 or Ag8653 cell, YO or YB2.0.
[0232] The invention further relates to a protein of interest,
preferably an antibody produced by any of the methods
described.
[0233] The invention further relates to a pharmaceutical
composition comprising a polynucleotide sequence useful for
blocking or reducing the expression of a protein having an amino
acid sequence comprising a steroidogenic acute regulatory related
lipid transfer (START) domain or a derivative or mutant thereof.
The invention furthermore relates to a pharmaceutical composition
comprising a polynucleotide sequence which blocks or reduces the
expression of a protein having an amino acid sequence comprising a
START domain or a derivative or mutant thereof.
[0234] In a specific embodiment of the present invention the
pharmaceutical composition is characterized in that the START
domain sequence is ceramide transfer protein CERT (CERT=SEQ ID NO.
11 or CERT.sub.L=SEQ ID NO. 13) or a derivative or mutant
thereof.
[0235] In another specific embodiment of the present invention the
pharmaceutical composition is characterized in that the START
domain is (SEQ ID NO. 17 or 19) or a derivative or mutant
thereof.
[0236] In a specific embodiment of the present invention the
pharmaceutical composition is characterized in that the
polynucleotide sequence is RNAi, siRNA or antisense-RNA.
[0237] In a preferred embodiment of the present invention the
pharmaceutical composition is characterized in that the START
domain protein is a mammalian START domain family member such as
PCTP (SEQ ID NO. 27), StarD7, GPBP, StarD10, StarD8, StarD13,
DLC-1, StarD4 (SEQ ID NO. 21), StarD6 (SEQ ID NO. 25), StarD5 (SEQ
ID NO. 23), MLN64, StAR, THEA-2, CACH or StarD9 or a derivative or
mutant thereof.
[0238] In a particularly preferred embodiment of the present
invention the pharmaceutical composition is characterized in that
said polynucleotide is complementary to the CERT nucleotide
sequence or parts thereof, especially to the START domain.
[0239] In a most preferred embodiment of the present invention the
pharmaceutical composition is characterized in that said
polynucleotide binds to either the CERT gene or the CERT
promoter.
[0240] In a further embodiment of the present invention the
pharmaceutical composition is characterized in that said
polynucleotide is anti-sense oligonucleotide to the CERT gene or
parts thereof.
[0241] The invention further relates to a pharmaceutical
composition comprising an inhibitor or suppressor of a protein
having an amino acid sequence comprising a steroidogenic acute
regulatory related lipid transfer (START) domain, preferably CERT
(SEQ ID NO. 11 or SEQ ID NO. 13) or a derivative or mutant
thereof.
[0242] In a specific embodiment of the present invention the
pharmaceutical composition is characterized in that said inhibitor
or suppressor is a chemical substance or a peptid-inhibitor or an
inhibiting protein such as. (i) protein binding to CERT promoter
thereby inhibiting CERT expression, (ii) protein binding to CERT or
PKD thus preventing binding of PKD and CERT and hindering CERT
phosphorylation by PKD, (iii) a protein similar to CERT which
however does not fulfill CERT functions, that means a
"dominant-negative" CERT variant, or (iv) a protein acting as
scaffold for both CERT and PKD, resulting in irreversible binding
of CERT to PKD (=a stable PKD/CERT complex) which is not functional
due to the inhibitory phosphorylation of CERT by PKD and the
hindering of dissociation of CERT from said complex.
[0243] In a specific embodiment of the present invention the
pharmaceutical composition is is characterized in that said
inhibitor or suppressor is a inhibitor or suppressor of CERT
activity.
[0244] The invention further relates to a method for identifying a
modulator of START domain protein function, preferably CERT
function, comprising providing a protein having an amino acid
sequence comprising a steroidogenic acute regulatory related lipid
transfer (START) domain or a derivative or mutant thereof,
preferably CERT, contacting said protein of step a) with a test
agent, determining an effect related to increased or decreased
protein secretion or expression of cell-surface proteins.
[0245] The invention further relates to a method comprising
application of a pharmaceutical composition as described for the
treatment of cancer.
[0246] The invention furthermore relates to a use of a START domain
protein or a polynucleotide encoding for a START domain protein to
increase secretion and/or production of a protein of interest.
[0247] The invention further relates to a diagnostic use of any of
the methods, expression vectors, cells or pharmaceutical
compositions as described.
[0248] In a specific embodiment the invention further relates to a
method of producing a heterologous protein of interest in a cell
comprising increasing the expression or activity of a protein
having an amino acid sequence comprising a steroidogenic acute
regulatory related lipid transfer (START) domain consensus sequence
or a derivative or mutant thereof as listed below,
TABLE-US-00002 CONSENSUS/80% (SEQ ID NO 28)
nhnntnnntnhtnhhntnnnWnnnnnnnnnnnnnnnnnhhthnnnnnnnn nnnnnnnnnnn +
hnthhnnnnnnnhnnnhhntnnnnnntWppnhnnnn
nnnnnnnnnhthlpnhtnsnnnnnnnsnlnhnnntnnhnnnhnsnR-hhn
lRnhpnnnnnnnnnnnttnhhlhnnohpnntnnnnnnnnnthhRsphhns
hhhhpnnttsnnnnnnnnnnnnsphhhlnnh-htsnnnnnnnpnhhpnhh
tnthnnhhpnnnnhtthptntnp
[0249] Whereby the class key residues are (represented in the one
letter amino acid code):
TABLE-US-00003 alcohol o S, T aliphatic l I, L, V any n A, C, D, E,
F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y aromatic a F, H, W,
Y charged c D, E, H, K, R hydrophobic h A, C, F, G, H, I, K, L, M,
R, T, V, W, Y negative - D, E polar p C, D, E, H, K, N, Q, R, S, T
positive + H, K, R small s A, C, D, G, N, P, S, T, V tiny u A, G, S
turnlike t A, C, D, E, G, H, K, N, Q, R, S, T
and effecting the expression of said protein of interest.
[0250] In further preferred embodiments of the invention the
protein having an amino acid sequence comprising a steroidogenic
acute regulatory related lipid transfer (START) domain in any of
the previous embodiments (e.g. expression vectors, cells, proteins,
pharmaceutical compositions, methods and uses) is defined by
comprising a START domain consensus sequence or a derivative or
mutant thereof as listed above (SEQ ID NO 28; see also FIG. 9).
[0251] The invention generally described above will be more readily
understood by reference to the is following examples, which are
hereby included merely for the purpose of illustration of certain
embodiments of the present invention. The following examples are
not limiting. They merely show possible embodiments of the
invention. A person skilled in the art could easily adjust the
conditions to apply it to other embodiments.
EXPERIMENTAL
Materials and Methods
Antibodies and Reagents
[0252] Antibodies are: rabbit anti-PKD substrate polyclonal
antibody (Cell Signaling), mouse anti-Flag monoclonal antibody
(Sigma-Aldrich), mouse anti-GFP monoclonal antibody (Roche), rabbit
anti-PKD polyclonal antibody (C-20, Santa Cruz Biotechnology),
mouse anti-GS28 (BD Biosciences) and mouse anti-tubulin
(Neomarkers). The phosphospecific anti-pS916 PKD antibody
monitoring PKD autophosphorylation is described elsewhere (Hausser
et al., 2002). Peroxidase-labeled secondary anti-mouse and
anti-rabbit IgG antibodies are from Amersham; alkaline
phosphatase-labeled secondary anti-mouse IgG antibody is from
Sigma; Alexa Fluor 488- and 546-labeled secondary anti-mouse and
anti-rat IgG antibodies are from Molecular Probes.
DNA Constructs
[0253] Full-length CERT cDNA is amplified by PCR using
pcDNA3-Flag-CERT as a template with primers containing EcoRI
restriction sites and cloned into the pEGFPC1 vector. The point
mutants of CERT are generated by Quikchange site-directed PCR
mutagenesis following the manufacturer's instructions (Stratagene).
Truncated CERT variants are generated by insertion of STOP codons.
The following oligonucleotides are used: CERT-S132A (SEQ ID NO.1:
5'-cgtcgacatggcgcaatggtgtccctgg-3'), CERT-S132A rev (SEQ ID NO.2:
5'-ccagggacaccattgcgccatgtcgacg-3'), CERT-S272A (SEQ ID NO.3:
5'-ggttaaacgtgaggacgcctggcagaagagactgg-3'); CERT-S272Arev (SEQ ID
NO.4: 5'-ccagtctcttctgccaggcgtcctcacgtttaacc-3'), CERT truncations
at amino acid 138 (SEQ ID NO.5:
5'-ggtgtccctggtgtcttgagcaagtggctactc-3'); CERT-138 truncation rev
(SEQ ID NO.6: 5'-gagtagccacttgctcaagacaccagggacacc-3'). The
Flag-CERT cDNA is subcloned into pGEX6P1 using EcoRI restriction
sites. pEGFP-N-1-PKD and pEGFP-N1-PKD.sub.K612W are described
previously (Hausser et al., 2005). The plasmid encoding ss-HRP-Flag
is kindly provided by Vivek Malhotra (UCSD).
cDNAs and Proteins
TABLE-US-00004 human: CERT cDNA (SEQ ID NO. 10): atgtcggata
atcagagctg gaactcgtcg ggctcggagg aggatccaga gacggagtct 60
gggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacat tcatgggtgg
120 caggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatc
tgaagatgaa 180 acagagtatg gctgcagagg atccatctgt cttagcaagg
ctgtcatcac acctcacgat 240 tttgatgaat gtcgatttga tattagtgta
aatgatagtg tttggtatct tcgtgctcag 300 gatccagatc atagacagca
atggatagat gccattgaac agcacaagac tgaatctgga 360 tatggatctg
aatccagctt gcgtcgacat ggctcaatgg tgtccctggt gtctggagca 420
agtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagttt acgtgagaag
480 ttggctgaaa tggaaacatt tagagacatc ttatgtagac aagttgacac
gctacagaag 540 tactttgatg cctgtgctga tgctgtctct aaggatgaac
ttcaaaggga taaagtggta 600 gaagatgatg aagatgactt tcctacaacg
cgttctgatg gtgacttctt gcatagtacc 660 aacggcaata aagaaaagtt
atttccacat gtgacaccaa aaggaattaa tggtatagac 720 tttaaagggg
aagcgataac ttttaaagca actactgctg gaatccttgc aacactttct 780
cattgtattg aactaatggt taaacgtgag gacagctggc agaagagact ggataaggaa
840 actgagaaga aaagaagaac agaggaagca tataaaaatg caatgacaga
acttaagaaa 900 aaatcccact ttggaggacc agattatgaa gaaggcccta
acagtctgat taatgaagaa 960 gagttctttg atgctgttga agctgctctt
gacagacaag ataaaataga agaacagtca 1020 cagagtgaaa aggtgagatt
acattggcct acatccttgc cctctggaga tgccttttct 1080 tctgtgggga
cacatagatt tgtccaaaag gttgaagaga tggtgcagaa ccacatgact 1140
tactcattac aggatgtagg cggagatgcc aattggcagt tggttgtaga agaaggagaa
1200 atgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcc
tttaaaagct 1260 acccatgcag ttaaaggcgt cacaggacat gaagtctgca
attatttctg gaatgttgac 1320 gttcgcaatg actgggaaac aactatagaa
aactttcatg tggtggaaac attagctgat 1380 aatgcaatca tcatttatca
aacacacaag agggtgtggc ctgcttctca gcgagacgta 1440 ttatatcttt
ctgtcattcg aaagatacca gccttgactg aaaatgaccc tgaaacttgg 1500
atagtttgta atttttctgt ggatcatgac agtgctcctc taaacaaccg atgtgtccgt
1560 gccaaaataa atgttgctat gatttgtcaa accttggtaa gcccaccaga
gggaaaccag 1620 gaaattagca gggacaacat tctatgcaag attacatatg
tagctaatgt gaaccctgga 1680 ggatgggcac cagcctcagt gttaagggca
gtggcaaagc gagagtatcc taaatttcta 1740 aaacgtttta cttcttacgt
ccaagaaaaa actgcaggaa agcctatttt gttctag 1797 human: CERT protein
(SEQ ID NO 11) Met Ser Asp Asn Gln Ser Trp Asn Ser Ser Gly Ser Glu
Glu Asp Pro Glu Thr Glu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu
Ser Lys Trp Thr Asn Tyr Ile His Gly Trp Gln Asp Arg Trp Val Val Leu
Lys Asn Asn Ala Leu Ser Tyr Tyr Lys Ser Glu Asp Glu Thr Glu Tyr Gly
Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala Val Ile Thr Pro His Asp Phe
Asp Glu Cys Arg Phe Asp Ile Ser Val Asn Asp Ser Val Trp Tyr Leu Arg
Ala Gln Asp Pro Asp His Arg Gln Gln Trp Ile Asp Ala Ile Glu Gln His
Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg Arg His Gly Ser
Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser Ala Thr Ser Thr Ser
Ser Phe Lys Lys Gly His Ser Leu Arg Glu Lys Leu Ala Glu Met Glu Thr
Phe Arg Asp Ile Leu Cys Arg Gln Val Asp Thr Leu Gln Lys Tyr Phe Asp
Ala Cys Ala Asp Ala Val Ser Lys Asp Glu Leu Gln Arg Asp Lys Val Val
Glu Asp Asp Glu Asp Asp Phe Pro Thr Thr Arg Ser Asp Gly Asp Phe Leu
His Ser Thr Asn Gly Asn Lys Glu Lys Leu Phe Pro His Val Thr Pro Lys
Gly Ile Asn Gly Ile Asp Phe Lys Gly Glu Ala Ile Thr Phe Lys Ala Thr
Thr Ala Gly Ile Leu Ala Thr Leu Ser His Cys Ile Glu Leu Met Val Lys
Arg Glu Asp Ser Trp Gln Lys Arg Leu Asp Lys Glu Thr Glu Lys Lys Arg
Arg Thr Glu Glu Ala Tyr Lys Asn Ala Met Thr Glu Leu Lys Lys Lys Ser
His Phe Gly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Ser Leu Ile Asn Glu
Glu Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gln Asp Lys Ile
Glu Glu Gln Ser Gln Ser Glu Lys Val Arg Leu His Trp Pro Thr Ser Leu
Pro Ser Gly Asp Ala Phe Ser Ser Val Gly Thr His Arg Phe Val Gln Lys
Val Glu Glu Met Val Gln Asn His Met Thr Tyr Ser Leu Gln Asp Val Gly
Gly Asp Ala Asn Trp Gln Leu Val Val Glu Glu Gly Glu Met Lys Val Tyr
Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu Asp Pro Leu Lys Ala Thr
His Ala Val Lys Gly Val Thr Gly His Glu Val Cys Asn Tyr Phe Trp Asn
Val Asp Val Arg Asn Asp Trp Glu Thr Thr Ile Glu Asn Phe His Val Val
Glu Thr Leu Ala Asp Asn Ala Ile Ile Ile Tyr Gln Thr His Lys Arg Val
Trp Pro Ala Ser Gln Arg Asp Val Leu Tyr Leu Ser Val Ile Arg Lys Ile
Pro Ala Leu Thr Glu Asn Asp Pro Glu Thr Trp Ile Val Cys Asn Phe Ser
Val Asp His Asp Ser Ala Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile
Asn Val Ala Met Ile Cys Gln Thr Leu Val Ser Pro Pro Glu Gly Asn Gln
Glu Ile Ser Arg Asp Asn Ile Leu Cys Lys Ile Thr Tyr Val Ala Asn Val
Asn Pro Gly Gly Trp Ala Pro Ala Ser Val Leu Arg Ala Val Ala Lys Arg
Glu Tyr Pro Lys Phe Leu Lys Arg Phe Thr Ser Tyr Val Gln Glu Lys Thr
Ala Gly Lys Pro Ile Leu Phe human: CERT L cDNA (SEQ ID NO 12)
gcaggaagat ggcggcggta gcggaggtgt gagtggacgc gggactcagc ggccggattt
60 tctcttccct tcttttccct tttccttccc tatttgaaat tggcatcgag
ggggctaagt 120 tcgggtggca gcgccgggcg caacgcaggg gtcacggcga
cggcggcggc ggctgacggc 180 tggaagggta ggcttcattc accgctcgtc
ctccttcctc gctccgctcg gtgtcaggcg 240 cggcggcggc gcggcgggcg
gacttcgtcc ctcctcctgc tcccccccac accggagcgg 300 gcactcttcg
cttcgccatc ccccgaccct tcaccccgag gactgggcgc ctcctccggc 360
gcagctgagg gagcgggggc cggtctcctg ctcggttgtc gagcctccat gtcggataat
420 cagagctgga actcgtcggg ctcggaggag gatccagaga cggagtctgg
gccgcctgtg 480 gagcgctgcg gggtcctcag taagtggaca aactacattc
atgggtggca ggatcgttgg 540 gtagttttga aaaataatgc tctgagttac
tacaaatctg aagatgaaac agagtatggc 600 tgcagaggat ccatctgtct
tagcaaggct gtcatcacac ctcacgattt tgatgaatgt 660 cgatttgata
ttagtgtaaa tgatagtgtt tggtatcttc gtgctcagga tccagatcat 720
agacagcaat ggatagatgc cattgaacag cacaagactg aatctggata tggatctgaa
780 tccagcttgc gtcgacatgg ctcaatggtg tccctggtgt ctggagcaag
tggctactct 840 gcaacatcca cctcttcatt caagaaaggc cacagtttac
gtgagaagtt ggctgaaatg 900 gaaacattta gagacatctt atgtagacaa
gttgacacgc tacagaagta ctttgatgcc 960 tgtgctgatg ctgtctctaa
ggatgaactt caaagggata aagtggtaga agatgatgaa 1020 gatgactttc
ctacaacgcg ttctgatggt gacttcttgc atagtaccaa cggcaataaa 1080
gaaaagttat ttccacatgt gacaccaaaa ggaattaatg gtatagactt taaaggggaa
1140 gcgataactt ttaaagcaac tactgctgga atccttgcaa cactttctca
ttgtattgaa 1200 ctaatggtta aacgtgagga cagctggcag aagagactgg
ataaggaaac tgagaagaaa 1260 agaagaacag aggaagcata taaaaatgca
atgacagaac ttaagaaaaa atcccacttt 1320 ggaggaccag attatgaaga
aggccctaac agtctgatta atgaagaaga gttctttgat 1380 gctgttgaag
ctgctcttga cagacaagat aaaatagaag aacagtcaca gagtgaaaag 1440
gtgagattac attggcctac atccttgccc tctggagatg ccttttcttc tgtggggaca
1500 catagatttg tccaaaagcc ctatagtcgc tcttcctcca tgtcttccat
tgatctagtc 1560 agtgcctctg atgatgttca cagattcagc tcccaggttg
aagagatggt gcagaaccac 1620 atgacttact cattacagga tgtaggcgga
gatgccaatt ggcagttggt tgtagaagaa 1680 ggagaaatga aggtatacag
aagagaagta gaagaaaatg ggattgttct ggatccttta 1740 aaagctaccc
atgcagttaa aggcgtcaca ggacatgaag tctgcaatta tttctggaat 1800
gttgacgttc gcaatgactg ggaaacaact atagaaaact ttcatgtggt ggaaacatta
1860 gctgataatg caatcatcat ttatcaaaca cacaagaggg tgtggcctgc
ttctcagcga 1920 gacgtattat atctttctgt cattcgaaag ataccagcct
tgactgaaaa tgaccctgaa 1980 acttggatag tttgtaattt ttctgtggat
catgacagtg ctcctctaaa caaccgatgt 2040 gtccgtgcca aaataaatgt
tgctatgatt tgtcaaacct tggtaagccc accagaggga 2100 aaccaggaaa
ttagcaggga caacattcta tgcaagatta catatgtagc taatgtgaac 2160
cctggaggat gggcaccagc ctcagtgtta agggcagtgg caaagcgaga gtatcctaaa
2220 tttctaaaac gttttacttc ttacgtccaa gaaaaaactg caggaaagcc
tattttgttc 2280 tagtattaac aggtactaga agatatgttt tatctttttt
taactttatt tgactaatat 2340 gactgtcaat actaaaattt agttgttgaa
agtatttact atgtttttt 2389 human: CERT L protein (SEQ ID NO 13) Met
Ser Asp Asn Gln Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp Pro Glu Thr
Glu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser Lys Trp Thr Asn
Tyr Ile His Gly Trp Gln Asp Arg Trp Val Val Leu Lys Asn Asn Ala Leu
Ser Tyr Tyr Lys Ser Glu Asp Glu Thr Glu Tyr Gly Cys Arg Gly Ser Ile
Cys Leu Ser Lys Ala Val Ile Thr Pro His Asp Phe Asp Glu Cys Arg Phe
Asp Ile Ser Val Asn Asp Ser Val Trp Tyr Leu Arg Ala Gln Asp Pro Asp
His Arg Gln Gln Trp Ile Asp Ala Ile Glu Gln His Lys Thr Glu Ser Gly
Tyr Gly Ser Glu Ser Ser Leu Arg Arg His Gly Ser Met Val Ser Leu Val
Ser Gly Ala Ser Gly Tyr Ser Ala Thr Ser Thr Ser Ser Phe Lys Lys Gly
His Ser Leu Arg Glu Lys Leu Ala Glu Met Glu Thr Phe Arg Asp Ile Leu
Cys Arg Gln Val Asp Thr Leu Gln Lys Tyr Phe Asp Ala Cys Ala Asp Ala
Val Ser Lys Asp Glu Leu Gln Arg Asp Lys Val Val Glu Asp Asp Glu Asp
Asp Phe Pro Thr Thr Arg Ser Asp Gly Asp Phe Leu His Ser Thr Asn Gly
Asn Lys Glu Lys Leu Phe Pro His Val Thr Pro Lys Gly Ile Asn Gly Ile
Asp
Phe Lys Gly Glu Ala Ile Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu Ala
Thr Leu Ser His Cys Ile Glu Leu Met Val Lys Arg Glu Asp Ser Trp Gln
Lys Arg Leu Asp Lys Glu Thr Glu Lys Lys Arg Arg Thr Glu Glu Ala Tyr
Lys Asn Ala Met Thr Glu Leu Lys Lys Lys Ser His Phe Gly Gly Pro Asp
Tyr Glu Glu Gly Pro Asn Ser Leu Ile Asn Glu Glu Glu Phe Phe Asp Ala
Val Glu Ala Ala Leu Asp Arg Gln Asp Lys Ile Glu Glu Gln Ser Gln Ser
Glu Lys Val Arg Leu His Trp Pro Thr Ser Leu Pro Ser Gly Asp Ala Phe
Ser Ser Val Gly Thr His Arg Phe Val Gln Lys Pro Tyr Ser Arg Ser Ser
Ser Met Ser Ser Ile Asp Leu Val Ser Ala Ser Asp Asp Val His Arg Phe
Ser Ser Gln Val Glu Glu Met Val Gln Asn His Met Thr Tyr Ser Leu Gln
Asp Val Gly Gly Asp Ala Asn Trp Gln Leu Val Val Glu Glu Gly Glu Met
Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu Asp Pro Leu
Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu Val Cys Asn Tyr
Phe Trp Asn Val Asp Val Arg Asn Asp Trp Glu Thr Thr Ile Glu Asn Phe
His Val Val Glu Thr Leu Ala Asp Asn Ala Ile Ile Ile Tyr Gln Thr His
Lys Arg Val Trp Pro Ala Ser Gln Arg Asp Val Leu Tyr Leu Ser Val Ile
Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp Pro Glu Thr Trp Ile Val Cys
Asn Phe Ser Val Asp His Asp Ser Ala Pro Leu Asn Asn Arg Cys Val Arg
Ala Lys Ile Asn Val Ala Met Ile Cys Gln Thr Leu Val Ser Pro Pro Glu
Gly Asn Gln Glu Ile Ser Arg Asp Asn Ile Leu Cys Lys Ile Thr Tyr Val
Ala Asn Val Asn Pro Gly Gly Trp Ala Pro Ala Ser Val Leu Arg Ala Val
Ala Lys Arg Glu Tyr Pro Lys Phe Leu Lys Arg Phe Thr Ser Tyr Val Gln
Glu Lys Thr Ala Gly Lys Pro Ile Leu Phe human: CERT S132A cDNA (SEQ
ID NO 14) atgtcggata atcagagctg gaactcgtcg ggctcggagg aggatccaga
gacggagtct 60 gggccgcctg tggagcgctg cggggtcctc agtaagtgga
caaactacat tcatgggtgg 120 caggatcgtt gggtagtttt gaaaaataat
gctctgagtt actacaaatc tgaagatgaa 180 acagagtatg gctgcagagg
atccatctgt cttagcaagg ctgtcatcac acctcacgat 240 tttgatgaat
gtcgatttga tattagtgta aatgatagtg tttggtatct tcgtgctcag 300
gatccagatc atagacagca atggatagat gccattgaac agcacaagac tgaatctgga
360 tatggatctg aatccagctt gcgtcgacat ggcgcaatgg tgtccctggt
gtctggagca 420 agtggctact ctgcaacatc cacctcttca ttcaagaaag
gccacagttt acgtgagaag 480 ttggctgaaa tggaaacatt tagagacatc
ttatgtagac aagttgacac gctacagaag 540 tactttgatg cctgtgctga
tgctgtctct aaggatgaac ttcaaaggga taaagtggta 600 gaagatgatg
aagatgactt tcctacaacg cgttctgatg gtgacttctt gcatagtacc 660
aacggcaata aagaaaagtt atttccacat gtgacaccaa aaggaattaa tggtatagac
720 tttaaagggg aagcgataac ttttaaagca actactgctg gaatccttgc
aacactttct 780 cattgtattg aactaatggt taaacgtgag gacagctggc
agaagagact ggataaggaa 840 actgagaaga aaagaagaac agaggaagca
tataaaaatg caatgacaga acttaagaaa 900 aaatcccact ttggaggacc
agattatgaa gaaggcccta acagtctgat taatgaagaa 960 gagttctttg
atgctgttga agctgctctt gacagacaag ataaaataga agaacagtca 1020
cagagtgaaa aggtgagatt acattggcct acatccttgc cctctggaga tgccttttct
1080 tctgtgggga cacatagatt tgtccaaaag gttgaagaga tggtgcagaa
ccacatgact 1140 tactcattac aggatgtagg cggagatgcc aattggcagt
tggttgtaga agaaggagaa 1200 atgaaggtat acagaagaga agtagaagaa
aatgggattg ttctggatcc tttaaaagct 1260 acccatgcag ttaaaggcgt
cacaggacat gaagtctgca attatttctg gaatgttgac 1320 gttcgcaatg
actgggaaac aactatagaa aactttcatg tggtggaaac attagctgat 1380
aatgcaatca tcatttatca aacacacaag agggtgtggc ctgcttctca gcgagacgta
1440 ttatatcttt ctgtcattcg aaagatacca gccttgactg aaaatgaccc
tgaaacttgg 1500 atagtttgta atttttctgt ggatcatgac agtgctcctc
taaacaaccg atgtgtccgt 1560 gccaaaataa atgttgctat gatttgtcaa
accttggtaa gcccaccaga gggaaaccag 1620 gaaattagca gggacaacat
tctatgcaag attacatatg tagctaatgt gaaccctgga 1680 ggatgggcac
cagcctcagt gttaagggca gtggcaaagc gagagtatcc taaatttcta 1740
aaacgtttta cttcttacgt ccaagaaaaa actgcaggaa agcctatttt gttctag 1797
human: CERT S132A protein (SEQ ID NO 15) Met Ser Asp Asn Gln Ser
Trp Asn Ser Ser Gly Ser Glu Glu Asp Pro Glu Thr Glu Ser Gly Pro Pro
Val Glu Arg Cys Gly Val Leu Ser Lys Trp Thr Asn Tyr Ile His Gly Trp
Gln Asp Arg Trp Val Val Leu Lys Asn Asn Ala Leu Ser Tyr Tyr Lys Ser
Glu Asp Glu Thr Glu Tyr Gly Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala
Val Ile Thr Pro His Asp Phe Asp Glu Cys Arg Phe Asp Ile Ser Val Asn
Asp Ser Val Trp Tyr Leu Arg Ala Gln Asp Pro Asp His Arg Gln Gln Trp
Ile Asp Ala Ile Glu Gln His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser
Ser Leu Arg Arg His Gly Ala Met Val Ser Leu Val Ser Gly Ala Ser Gly
Tyr Ser Ala Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg Glu
Lys Leu Ala Glu Met Glu Thr Phe Arg Asp Ile Leu Cys Arg Gln Val Asp
Thr Leu Gln Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys Asp Glu
Leu Gln Arg Asp Lys Val Val Glu Asp Asp Glu Asp Asp Phe Pro Thr Thr
Arg Ser Asp Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys Glu Lys Leu
Phe Pro His Val Thr Pro Lys Gly Ile Asn Gly Ile Asp Phe Lys Gly Glu
Ala Ile Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu Ala Thr Leu Ser His
Cys Ile Glu Leu Met Val Lys Arg Glu Asp Ser Trp Gln Lys Arg Leu Asp
Lys Glu Thr Glu Lys Lys Arg Arg Thr Glu Glu Ala Tyr Lys Asn Ala Met
Thr Glu Leu Lys Lys Lys Ser His Phe Gly Gly Pro Asp Tyr Glu Glu Gly
Pro Asn Ser Leu Ile Asn Glu Glu Glu Phe Phe Asp Ala Val Glu Ala Ala
Leu Asp Arg Gln Asp Lys Ile Glu Glu Gln Ser Gln Ser Glu Lys Val Arg
Leu His Trp Pro Thr Ser Leu Pro Ser Gly Asp Ala Phe Ser Ser Val Gly
Thr His Arg Phe Val Gln Lys Val Glu Glu Met Val Gln Asn His Met Thr
Tyr Ser Leu Gln Asp Val Gly Gly Asp Ala Asn Trp Gln Leu Val Val Glu
Glu Gly Glu Met Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val
Leu Asp Pro Leu Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu
Val Cys Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp Trp Glu Thr Thr
Ile Glu Asn Phe His Val Val Glu Thr Leu Ala Asp Asn Ala Ile Ile Ile
Tyr Gln Thr His Lys Arg Val Trp Pro Ala Ser Gln Arg Asp Val Leu Tyr
Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp Pro Glu Thr
Trp Ile Val Cys Asn Phe Ser Val Asp His Asp Ser Ala Pro Leu Asn Asn
Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile Cys Gln Thr Leu Val
Ser Pro Pro Glu Gly Asn Gln Glu Ile Ser Arg Asp Asn Ile Leu Cys Lys
Ile Thr Tyr Val Ala Asn Val Asn Pro Gly Gly Trp Ala Pro Ala Ser Val
Leu Arg Ala Val Ala Lys Arg Glu Tyr Pro Lys Phe Leu Lys Arg Phe Thr
Ser Tyr Val Gln Glu Lys Thr Ala Gly Lys Pro Ile Leu Phe human:
START Domain CERT cDNA (SEQ ID NO 16) agatttgtcc aaaaggttga
agagatggtg cagaaccaca tgacttactc attacaggat 60 gtaggcggag
atgccaattg gcagttggtt gtagaagaag gagaaatgaa ggtatacaga 120
agagaagtag aagaaaatgg gattgttctg gatcctttaa aagctaccca tgcagttaaa
180 ggcgtcacag gacatgaagt ctgcaattat ttctggaatg ttgacgttcg
caatgactgg 240 gaaacaacta tagaaaactt tcatgtggtg gaaacattag
ctgataatgc aatcatcatt 300 tatcaaacac acaagagggt gtggcctgct
tctcagcgag acgtattata tctttctgtc 360 attcgaaaga taccagcctt
gactgaaaat gaccctgaaa cttggatagt ttgtaatttt 420 tctgtggatc
atgacagtgc tcctctaaac aaccgatgtg tccgtgccaa aataaatgtt 480
gctatgattt gtcaaacctt ggtaagccca ccagagggaa accaggaaat tagcagggac
540 aacattctat gcaagattac atatgtagct aatgtgaacc ctggaggatg
ggcaccagcc 600 tcagtgttaa gggcagtggc aaagcgagag tatcctaaat
ttctaaaacg ttttacttct 660 tacgtccaa 669 human: START Domain CERT
protein (SEQ ID NO 17) Arg Phe Val Gln Lys Val Glu Glu Met Val Gln
Asn His Met Thr Tyr Ser Leu Gln Asp Val Gly Gly Asp Ala Asn Trp Gln
Leu Val Val Glu Glu Gly Glu Met Lys Val Tyr Arg Arg Glu Val Glu Glu
Asn Gly Ile Val Leu Asp Pro Leu Lys Ala Thr His Ala Val Lys Gly Val
Thr Gly His Glu Val Cys Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp
Trp Glu Thr Thr Ile Glu Asn Phe His Val Val Glu Thr Leu Ala Asp Asn
Ala Ile Ile Ile Tyr Gln Thr His Lys Arg Val Trp Pro Ala Ser Gln Arg
Asp Val Leu Tyr Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr Glu Asn
Asp Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val Asp His Asp Ser Ala
Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile Cys
Gln Thr Leu Val Ser Pro Pro Glu Gly Asn Gln Glu Ile Ser Arg Asp Asn
Ile Leu Cys Lys Ile Thr Tyr Val Ala Asn Val Asn Pro Gly Gly Trp Ala
Pro Ala Ser Val Leu Arg Ala Val Ala Lys Arg Glu Tyr Pro Lys Phe Leu
Lys Arg Phe Thr Ser Tyr Val Gln human: START Domain CERT L cDNA
(SEQ ID NO 18) caggttgaag agatggtgca gaaccacatg acttactcat
tacaggatgt aggcggagat 60 gccaattggc agttggttgt agaagaagga
gaaatgaagg tatacagaag agaagtagaa 120 gaaaatggga ttgttctgga
tcctttaaaa gctacccatg cagttaaagg cgtcacagga 180 catgaagtct
gcaattattt ctggaatgtt gacgttcgca atgactggga aacaactata 240
gaaaactttc atgtggtgga aacattagct gataatgcaa tcatcattta tcaaacacac
300
aagagggtgt ggcctgcttc tcagcgagac gtattatatc tttctgtcat tcgaaagata
360 ccagccttga ctgaaaatga ccctgaaact tggatagttt gtaatttttc
tgtggatcat 420 gacagtgctc ctctaaacaa ccgatgtgtc cgtgccaaaa
taaatgttgc tatgatttgt 480 caaaccttgg taagcccacc agagggaaac
caggaaatta gcagggacaa cattctatgc 540 aagattacat atgtagctaa
tgtgaaccct ggaggatggg caccagcctc agtgttaagg 600 gcagtggcaa
agcgagagta tcctaaattt ctaaaacgtt ttacttctta cgtccaag 658 human:
START Domain CERT L protein (SEQ ID NO 19) Gln Val Glu Glu Met Val
Gln Asn His Met Thr Tyr Ser Leu Gln Asp Val Gly Gly Asp Ala Asn Trp
Gln Leu Val Val Glu Glu Gly Glu Met Lys Val Tyr Arg Arg Glu Val Glu
Glu Asn Gly Ile Val Leu Asp Pro Leu Lys Ala Thr His Ala Val Lys Gly
Val Thr Gly His Glu Val Cys Asn Tyr Phe Trp Asn Val Asp Val Arg Asn
Asp Trp Glu Thr Thr Ile Glu Asn Phe His Val Val Glu Thr Leu Ala Asp
Asn Ala Ile Ile Ile Tyr Gln Thr His Lys Arg Val Trp Pro Ala Ser Gln
Arg Asp Val Leu Tyr Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr Glu
Asn Asp Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val Asp His Asp Ser
Ala Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile
Cys Gln Thr Leu Val Ser Pro Pro Glu Gly Asn Gln Glu Ile Ser Arg Asp
Asn Ile Leu Cys Lys Ile Thr Tyr Val Ala Asn Val Asn Pro Gly Gly Trp
Ala Pro Ala Ser Val Leu Arg Ala Val Ala Lys Arg Glu Tyr Pro Lys Phe
Leu Lys Arg Phe Thr Ser Tyr Val Gln human: StarD4 cDNA (SEQ ID NO
20) actgttgaga gcggtgtgag gtgcttggta gcgcgccgta gctgcttcca
cgtccttgct 60 tcacctcagg taaagagaga agtaatggaa ggcctgtctg
atgttgcttc ttttgcaact 120 aaacttaaaa acactctcat ccagtaccat
agcattgaag aagataagtg gcgagttgct 180 aagaaaacga aagatgtaac
tgtttggaga aaaccctcag aagaatttaa tggatatctc 240 tacaaagccc
aaggtgttat agatgacctt gtctatagta taatagacca tatacgccca 300
gggccttgtc gtttggattg ggacagcttg atgacttctt tggatattct ggagaacttt
360 gaagagaatt gctgtgtgat gcgttacact actgctggtc agctttggaa
tataatttcc 420 ccaagagaat ttgttgattt ctcctatact gtgggctata
aagaagggct tttatcttgt 480 ggaataagtc ttgactggga tgaaaagaga
ccagaatttg ttcgaggata taaccatccc 540 tgtggttggt tttgtgttcc
acttaaagac aacccaaacc agagtctttt gacaggatat 600 attcagacag
atctgcgtgg gatgattcct cagtctgcgg tagatacagc catggcaagc 660
actttaacca acttctatgg tgatttacga aaagctttat gagaggcaaa atacattcaa
720 acttgtagta ctacagatca actctctcag ctacatggcc tgtaaaaatc
attgattcca 780 cttttctgca tagccggtag aaaaatttga aatgtttttg
gttcactagt acaatgtttg 840 gttttattcc taaagtaaat agctatctaa
gagagggcat tttcactttt ttttttttaa 900 attttgagac aggctctcac
tctgttgccc atgctggagg gcagtggtat gatcacagct 960 cactgcagct
ttgatctgac cgctcaaggg gttattctac ctcagcctcc tgaatagctg 1020
ggaatacagg tgcacgccac tatgcatggc taatttttgt ttaatttttt gtagagatgt
1080 ggtcacactg tgttgcccag gctggtcttg aactcctggc ctcaagtcat
tccccacctt 1140 agcctcccaa agtgttggga ttataagcgt gagccaccat
gcctggcccc aatttaaaat 1200 gtggaattca gttggtgtcc aagacttatc
ttgagactct taaaagcatc agtctgtaac 1260 tagaacaaat acagtcttag
atttacccaa gtgcctagat atcattttat aatgattaga 1320 attgagtatt
gtgggtcccc taattctgtg ggtgccttaa gtgagaattt ctaaatgatt 1380
ttcacattct aaatgacttt gggttttgaa ctctccatct agtttacttc taaaatggga
1440 acttgaggca attcaggtat ccaggcaaat ctttgtatat atttttttgt
gtacatgcac 1500 acatctcgaa atccatttcc gtgtttaatg ttagttgttt
atgtgttagt attcctgtgt 1560 ctactgtttt gttgttgtta atatgggtaa
agtgagccct gaaatacatg ctaaacaaga 1620 catgaaattc agaaaggtac
atagtgtttc aagtgcatgg tagtttgatc tgtgttttac 1680 tttattgtgt
tttcttgagt gtaaagaaag aataaatcaa agttcttcat acccattttg 1740
acaaagtgga acagtggagc tgttttttgc ttttgttttt atttattttt tgccactggt
1800 gatgatagat ttcaaaaaac aaaaggtggc agcagcacaa tgttcatggt
gaattatctc 1860 atagtatcta gattgatcaa gatctgacag aaggaatgca
caaaggattc tatattctta 1920 atgatttatt aattaccagg atccttttct
aaattgaatg tacttttgaa ttactaggtt 1980 tcttcttttt ttttgttctg
caatagtgaa agaaaactca gtagtttagt ttcagtttct 2040 catggaaatt
ggtaaatgtt agttttgact tcatctattt tttatttgtt tttattagcg 2100
tagagtagga agtctcatat tctactgttc tatctaggat ggtgaaattc caaaggtgcc
2160 taacttgagt aagggatttg tgacaagata gtacacatta ctataagggc
tattatttcc 2220 tgaactggat gtccctaaaa gcaaataaac tgcccactat ctct
2264 human: StarD4 protein (SEQ ID NO 21) Met Glu Gly Leu Ser Asp
Val Ala Ser Phe Ala Thr Lys Leu Lys Asn Thr Leu Ile Gln Tyr His Ser
Ile Glu Glu Asp Lys Trp Arg Val Ala Lys Lys Thr Lys Asp Val Thr Val
Trp Arg Lys Pro Ser Glu Glu Phe Asn Gly Tyr Leu Tyr Lys Ala Gln Gly
Val Ile Asp Asp Leu Val Tyr Ser Ile Ile Asp His Ile Arg Pro Gly Pro
Cys Arg Leu Asp Trp Asp Ser Leu Met Thr Ser Leu Asp Ile Leu Glu Asn
Phe Glu Glu Asn Cys Cys Val Met Arg Tyr Thr Thr Ala Gly Gln Leu Trp
Asn Ile Ile Ser Pro Arg Glu Phe Val Asp Phe Ser Tyr Thr Val Gly Tyr
Lys Glu Gly Leu Leu Ser Cys Gly Ile Ser Leu Asp Trp Asp Glu Lys Arg
Pro Glu Phe Val Arg Gly Tyr Asn His Pro Cys Gly Trp Phe Cys Val Pro
Leu Lys Asp Asn Pro Asn Gln Ser Leu Leu Thr Gly Tyr Ile Gln Thr Asp
Leu Arg Gly Met Ile Pro Gln Ser Ala Val Asp Thr Ala Met Ala Ser Thr
Leu Thr Asn Phe Tyr Gly Asp Leu Arg Lys Ala Leu human: StarD5 cDNA
(SEQ ID NO 22) gagctccagc ctccaggcac ccgggatcca gcgccgccgc
tcataacacc cgcgaccccg 60 cagctaagcg cagctcccga cgcaatggac
ccggcgctgg cagcccagat gagcgaggct 120 gtggccgaga agatgctcca
gtaccggcgg gacacagcag gctggaagat ttgccgggaa 180 ggcaatggag
tttcagtttc ctggaggcca tctgtggagt ttccagggaa cctgtaccga 240
ggagaaggca ttgtatatgg gacactagag gaggtgtggg actgtgtgaa gccagctgtt
300 ggaggcctac gagtgaagtg ggatgagaat gtgaccggtt ttgaaattat
ccaaagcatc 360 actgacaccc tgtgtgtaag cagaacctcc actccctccg
ctgccatgaa gctcatttct 420 cccagagatt ttgtggactt ggtgctagtc
aagagatatg aggatgggac catcagttcc 480 aacgccaccc atgtggagca
tccgttatgt cccccgaagc caggttttgt gagaggattt 540 aaccatcctt
gtggttgctt ctgtgaacct cttccagggg aacccaccaa gaccaacctg 600
gtcacattct tccataccga cctcagcggt tacctcccac agaacgtggt ggactccttc
660 ttcccccgca gcatgacccg gttttatgcc aaccttcaga aagcagtgaa
gcaattccat 720 gagtaatgct atcgttactt cttggcaaag aactcccgtg
actcatcgag gagctccagc 780 tgttgggaca ccaaggagcc tgggagcacg
cagaggcctg tgttcactct ttggaacaag 840 ctgatggact gcgcatctct
gagaatgcca accagaggcg gcagcccacc cttcctgcct 900 cctgccccac
tcagggttgg cgtgtgatga gccattcatg tgttccaaac tccatctgcc 960
tgttacccaa acacgcctct cctggcaggg tagacccagg cctctaacca tctgacagag
1020 actcggcctg gacaccatgc gatgcactct ggcaccaagg ctttatgtgc
ccatcactct 1080 cagagaccac gtttccctga ctgtcataga gaatcatcat
cgccactgaa aaccaggccc 1140 tgttgccttt taagcatgta ccgctccctc
agtcctgtgc tgcagccccc caaatatatt 1200 tttctgatat agaccttgta
tatggcttta atgccgcaaa atatttattt ttccttaaaa 1260 aaggtgtcaa
cttggaaata atggtttaaa aacaggataa gcattaagga aaaacaaaaa 1320
aaaaaaaaaa aaaaaaaaaa aaaa 1344 human: StarD5 protein (SEQ ID NO
23) Met Asp Pro Ala Leu Ala Ala Gln Met Ser Glu Ala Val Ala Glu Lys
Met Leu Gln Tyr Arg Arg Asp Thr Ala Gly Trp Lys Ile Cys Arg Glu Gly
Asn Gly Val Ser Val Ser Trp Arg Pro Ser Val Glu Phe Pro Gly Asn Leu
Tyr Arg Gly Glu Gly Ile Val Tyr Gly Thr Leu Glu Glu Val Trp Asp Cys
Val Lys Pro Ala Val Gly Gly Leu Arg Val Lys Trp Asp Glu Asn Val Thr
Gly Phe Glu Ile Ile Gln Ser Ile Thr Asp Thr Leu Cys Val Ser Arg Thr
Ser Thr Pro Ser Ala Ala Met Lys Leu Ile Ser Pro Arg Asp Phe Val Asp
Leu Val Leu Val Lys Arg Tyr Glu Asp Gly Thr Ile Ser Ser Asn Ala Thr
His Val Glu His Pro Leu Cys Pro Pro Lys Pro Gly Phe Val Arg Gly Phe
Asn His Pro Cys Gly Cys Phe Cys Glu Pro Leu Pro Gly Glu Pro Thr Lys
Thr Asn Leu Val Thr Phe Phe His Thr Asp Leu Ser Gly Tyr Leu Pro Gln
Asn Val Val Asp Ser Phe Phe Pro Arg Ser Met Thr Arg Phe Tyr Ala Asn
Leu Gln Lys Ala Val Lys Gln Phe His Glu human: StarD6 cDNA (SEQ ID
NO 24) atggacttca aggcaattgc ccaacaaact gcccaagaag ttttaggtta
taatcgagat 60 acatcaggct ggaaagtggt taaaacttca aaaaagataa
ctgtttccag taaggcttct 120 agaaaattcc atggaaatct atatcgtgtt
gaagggataa ttccagaatc accagctaaa 180 ctatctgatt tcctctacca
aactggagac agaattacat gggataaatc attgcaagtg 240 tataatatgg
tacacaggat tgattcggac acattcatat gtcataccat tacacaaagt 300
tttgccgtgg gctccatttc ccctcgagac tttatcgact tagtgtacat caagcgctac
360 gaaggaaata tgaacattat cagttctaaa agtgtggatt ttccagaata
tcctccatct 420 tcaaattata tccgcggtta taaccatcct tgtggctttg
tatgttcacc aatggaagaa 480 aacccagcat attccaaact agtgatgttt
gtccagacag aaatgagagg aaaattgtcc 540 ccatcaataa ttgaaaaaac
catgccttcc aacttagtaa acttcatcct caatgcaaaa 600 gatggaataa
aggcacacag aactccatca agacgtggat ttcatcataa tagtcattca 660 tga 663
human: StarD6 protein (SEQ ID NO 25) Met Asp Phe Lys Ala Ile Ala
Gln Gln Thr Ala Gln Glu Val Leu Gly Tyr Asn Arg Asp Thr Ser Gly Trp
Lys Val Val Lys Thr Ser Lys Lys
Ile Thr Val Ser Ser Lys Ala Ser Arg Lys Phe His Gly Asn Leu Tyr Arg
Val Glu Gly Ile Ile Pro Glu Ser Pro Ala Lys Leu Ser Asp Phe Leu Tyr
Gln Thr Gly Asp Arg Ile Thr Trp Asp Lys Ser Leu Gln Val Tyr Asn Met
Val His Arg Ile Asp Ser Asp Thr Phe Ile Cys His Thr Ile Thr Gln Ser
Phe Ala Val Gly Ser Ile Ser Pro Arg Asp Phe Ile Asp Leu Val Tyr Ile
Lys Arg Tyr Glu Gly Asn Met Asn Ile Ile Ser Ser Lys Ser Val Asp Phe
Pro Glu Tyr Pro Pro Ser Ser Asn Tyr Ile Arg Gly Tyr Asn His Pro Cys
Gly Phe Val Cys Ser Pro Met Glu Glu Asn Pro Ala Tyr Ser Lys Leu Val
Met Phe Val Gln Thr Glu Met Arg Gly Lys Leu Ser Pro Ser Ile Ile Glu
Lys Thr Met Pro Ser Asn Leu Val Asn Phe Ile Leu Asn Ala Lys Asp Gly
Ile Lys Ala His Arg Thr Pro Ser Arg Arg Gly Phe His His Asn Ser His
Ser human: PCTP cDNA (SEQ ID NO 26) ccggactgcg gaaggatgga
gctggccgcc ggaagcttct cggaggagca gttctgggag 60 gcctgcgccg
agctccagca gcccgctctg gccggggccg actggcagct cctagtggag 120
acctcgggca tcagcatcta ccggctgctg gacaagaaga ctggacttca tgagtataaa
180 gtctttggtg ttctggagga ctgctcacca actctactgg cagacatcta
tatggactca 240 gattacagaa aacaatggga ccagtatgtt aaagaactct
atgaacaaga atgcaacgga 300 gagactgtgg tctactggga agtgaagtac
ccttttccca tgtccaacag agactatgtc 360 taccttcggc agcggcgaga
cctggacatg gaagggagga agatccatgt gatcctggcc 420 cggagcacct
ccatgcctca gcttggcgag aggtctgggg tgatccgggt gaagcaatac 480
aagcagagcc tggcgattga gagtgacggc aagaagggga gcaaagtttt catgtattac
540 ttcgataacc cgggtggcca aattccgtcc tggctcatta actgggccgc
caagaatgga 600 gttcctaact tcttgaaaga catggcaaga gcctgtcaga
actacctcaa gaaaacctaa 660 gaaagagaac tgggaacatt gcatccatgg
gttgatgtct ctggaagtgc aaccacccaa 720 tgtctctgga agtgccacct
ggaagtgcca cctggaagtg tctctggaag agcacccacc 780 actgttcagc
cttcccctgc tgtttctgtc ttcagaggcc tacacactac cacatccttt 840
ctaagcatgt ttgcctgaca tccagctcac tcgtctgctt cctttctcgc tccccccatc
900 ctgggctggg ctgccttctt ctacagttca atatggggca gactagggaa
acctttgctt 960 gcttactatt aggaggggaa gtcttcagta gggaacacga
tcattccatt gtgcaatttt 1020 acggggatgg gtgggcggag ggacacaaca
aaatttaaga atgactattt gggcgggctg 1080 gctcttttgc agcttgtgat
ttcttccagc ttgggagggg ctgctggaag tggcatttcg 1140 ttcagagctg
actttcagtg cacccaaact ggatgacgtg ccaatgtcca tttgccttat 1200
gctttgtgga gctgattagg ctgggatttg aggtgataat ccagtaagtc tttcctcgtt
1260 cctacttgtg gaggatcagt agctgttatg atgccagacc atttggagaa
gtatcagagg 1320 cctgaccgga cacataatac gacaaccaca tttttcctca
tcatccatga ggaaatggat 1380 gatttctctt ttccatatgt cactggggga
aaggctgcct gtacctctca agctttgcat 1440 tttactggaa actgaggcgt
caagatggct gtggcagcta gcaaaagcaa agatgctttg 1500 tgcatagcct
tgtgaaaaag tatctttcta tgcaataaga tgaattttcc tcccagaata 1560
tttagaaatg tagaagggat aacagttcac agccaggtaa aatttaactg gtggcttaat
1620 gactctgcac ctttttctca ggaattctgc ctaagttgtc tgccttttct
accaccaaaa 1680 agacttttag ttttctatgc tttctcctga attttggtag
ggtaagtatt tctatgtcaa 1740 aggcacagcc ttgatgatct cagggaaaaa
ttttaatcac tgtgtataat gatactgaac 1800 cttgattaat aacagaaatt
caggatgtaa agccacagaa tgggatttat taatgtggga 1860 tacctcagac
tgtttgtttt ctttctggga agaaaagtgt gttctataat gaataaatat 1920
agagtggttt tt 1932 human: PCTP protein (SEQ ID NO 27) Met Glu Leu
Ala Ala Gly Ser Phe Ser Glu Glu Gln Phe Trp Glu Ala Cys Ala Glu Leu
Gln Gln Pro Ala Leu Ala Gly Ala Asp Trp Gln Leu Leu Val Glu Thr Ser
Gly Ile Ser Ile Tyr Arg Leu Leu Asp Lys Lys Thr Gly Leu His Glu Tyr
Lys Val Phe Gly Val Leu Glu Asp Cys Ser Pro Thr Leu Leu Ala Asp Ile
Tyr Met Asp Ser Asp Tyr Arg Lys Gln Trp Asp Gln Tyr Val Lys Glu Leu
Tyr Glu Gln Glu Cys Asn Gly Glu Thr Val Val Tyr Trp Glu Val Lys Tyr
Pro Phe Pro Met Ser Asn Arg Asp Tyr Val Tyr Leu Arg Gln Arg Arg Asp
Leu Asp Met Glu Gly Arg Lys Ile His Val Ile Leu Ala Arg Ser Thr Ser
Met Pro Gln Leu Gly Glu Arg Ser Gly Val Ile Arg Val Lys Gln Tyr Lys
Gln Ser Leu Ala Ile Glu Ser Asp Gly Lys Lys Gly Ser Lys Val Phe Met
Tyr Tyr Phe Asp Asn Pro Gly Gly Gln Ile Pro Ser Trp Leu Ile Asn Trp
Ala Ala Lys Asn Gly Val Pro Asn Phe Leu Lys Asp Met Ala Arg Ala Cys
Gln Asn Tyr Leu Lys Lys Thr
Cell Culture and Transfection
[0254] HEK293T and COS 7 cells grow in RPMI supplemented with 10%
fetal calf serum (FCS) in a humidified atmosphere containing 5%
CO.sub.2. HEK293T cells are transfected using TransIT293 reagent
(Minis) according to the manufacturer's instructions. For
immunofluorescence, COS 7 cells are grown on glass coverslips for
24 hours and transfected with Lipofectamine 2000 reagent
(Invitrogen).
[0255] CHO cells as well as CHO-derived cell lines producing human
serum albumine (HSA) or a human monoclonal IgG antibody are
cultivated in suspension in serum-free media in surface-aerated
T-flasks (Nunc, Denmark) in incubators (Thermo, Germany) or shake
flasks (Nunc, Denmark) at a temperature of 37.degree. C. and in an
atmosphere containing 5% CO.sub.2.
[0256] Seedstock cultures are subcultivated every 2-3 days with
seeding densities of 2-3E5 cells/mL. The cell concentration is
determined in all cultures by using a hemocytometer. Viability is
assessed by the trypan blue exclusion method. All CHO production
cells are cultured in BI-proprietary media and their composition
may not be revealed.
[0257] CHO-derived cells are transfected using Lipofectamine.TM.
and PLUS.TM. Reagents (both Invitrogen, Germany) according to the
guidelines provided by the manufacturer.
Fed-Batch Cultivation
[0258] Cells are seeded at 3E05 cells/ml into 125 ml shake flasks
in 30 ml of BI-proprietary production medium without antibiotics or
MTX (Sigma-Aldrich, Germany). The cultures are agitated at 120 rpm
in 37.degree. C. and 5% CO.sub.2 which is later reduced to 2% as
cell numbers increase. Culture parameters including pH, glucose and
lactate concentrations are determined daily and pH is adjusted to
pH 7.0 using NaCO.sub.3 as needed. BI-proprietary feed solution is
added every 24 hrs. Cell densities and viability are determined by
trypan-blue exclusion using an automated CEDEX cell quantification
system (Innovatis). Samples from the cell culture fluid are
collected at day 3, 5 and 7 and subjected to titer measurement by
ELISA.
ELISA
[0259] Quantification of IgG molecules in the supernatant of the
cell clones is performed via sandwich ELISA technology. ELISA
plates are coated using a goat anti-human IgG Fc-Fragment antibody
(Dianova, Germany) at 4.degree. C. over night. After washing and
blocking of the plates with 1% BSA is solution, the samples are
added and incubated for 1.5 hours. After washing, the detection
antibody (alkaline-phosphatase conjugated goat anti-human kappa
light chain antibody) is added and colorimetric detection is
performed by incubation with 4-nitrophenyl phosphate disodium salt
hexahydrate (Sigma, Germany) as substrate. After 20 min incubation
in the dark, the reaction is stopped and the absorbance is
immediately measured using an absorbance reader (Tecan, Germany)
with 405/492 nm. The concentration is calculated according to the
standard curve which is present on each plate.
[0260] Quantitative determination of secreted HSA in culture
samples is performed similarly, using the antibodies contained in
the Human Albumin ELISA Quantitation Kit (Bethyl Labs, Texas, USA)
and following the manufacturers instructions.
Immunofluorescence Microscopy
[0261] Cells are washed with PBS containing magnesium and calcium,
fixed in 4% paraformaldehyde at room temperature for 10 min, washed
and incubated with PBS containing 0.1 M glycine for 15 min. Cells
are then permeabilized with PBS containing 0.1% Triton for 5 min
and then blocked with 5% goat serum in PBS containing 0.1% Tween-20
for 30 min. Cells are incubated with primary antibody diluted in
blocking buffer for 2 hours, followed by incubation with secondary
antibodies diluted in blocking buffer for 1 hour. Coverslips are
mounted in Fluoromount G (Southern Biotechnology) and cells are
analyzed on a confocal laser scanning microscope (TCS SL, Leica)
using 488 and 543 nm excitation and a 40.0/1.25 HCX PL APO
objective lens. Images are processed with Adobe Photoshop.
Protein Extraction, Immunoprecipitation and Western Blotting
[0262] Whole cell extracts are obtained by solubilizing cells in
NP40 extraction buffer (NEB) [50 mM Tris (pH 7.5), 150 mM NaCl, 1%
NP40, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 20 mM
.beta.-glycerophosphate plus Complete protease inhibitors]. Lysates
are clarified by centrifugation at 16,000.times.g for 10 min. For
immunoprecipitations, equal amounts of protein are incubated with
specific antibodies for 2 h on ice. Immune complexes are collected
with protein G-Sepharose (GE Healthcare) and washed three times
with NEB (see above). Whole cell extracts or immunoprecipitated
proteins are boiled in sample buffer and subjected to SDS-PAGE. The
proteins are blotted onto polyvinylidine difluoride membranes
(Roth). After blocking with 0.5% blocking reagent (Roche) in PBS
containing 0.1% Tween 20, filters are probed with specific
antibodies. Proteins are visualized with peroxidase-coupled
secondary antibody using the enhanced chemiluminescence detection
system (Pierce). Stripping of membranes is performed in SDS buffer
[62.5 mM Tris (pH 6.8), 2% SDS, and 100 mM .beta.-mercaptoethanol]
for 30 min at 60.degree. C. Membranes are then reprobed with the
indicated antibodies.
Recombinant Protein Purification and In Vitro Kinase Assays
[0263] BL21 bacteria are transformed with pGEX6P-Flag-CERT(1-138)
and CERT-S132A(1-138) vectors. Expression is induced with 0.5 mM
isopropyl-.beta.-D-1-thiogalactopyranoside for 4 hrs at 30.degree.
C. Bacteria are harvested and resuspended in PBS containing 50
.mu.g/ml lysozyme, Complete protease inhibitors (Roche), 10 mM
sodium fluoride and 20 mM-glycerophosphate. Triton X-100 is added
to a final concentration of 1% prior to sonication. GST-CERT
fusions are purified from clarified lysate with glutathione resin
(GE Healthcare). The purity of protein preparations is verified by
SDS-PAGE and Coomassie staining Recombinant proteins are incubated
with is purified PKD1 in kinase buffer [50 mM Tris, pH 7.5, 10 mM
MgCl2 and 1 mM DTT] in the presence of either 2 .mu.Ci
[.gamma.-.sup.32P]-ATP or 75 .mu.M cold ATP for 30 min. Samples are
resolved by SDS-PAGE, blotted onto membrane and analyzed on a
PhosphoImager (Molecular Dynamics) or by Western blotting with
anti-PKD substrate antibody.
PIP Arrays
[0264] HEK293T cells transiently expressing GFP-tagged CERT
variants are harvested in hypotonic buffer [50 mM Tris, pH 7.4,
containing Complete protease inhibitors (Roche), 1 mM PMSF, 5 mM
.beta.-glycerophosphate and 5 mM sodium fluoride] and sheared by
passage through a 25G/16 mm gauge needle. The cytosol fraction is
obtained after 100,000.times.g centrifugation for 1 h and the
amount of expressed protein is quantified by measuring GFP peak
emission at 480-550 nm (excitation 466 nm). PIP arrays (Echelon)
are blocked in TBS-T [10 mM Tris, pH 8, 150 mM NaCl, 0.1% Tween-20]
containing 3% fatty acid-free BSA (Roth), followed by incubation
with 500 .mu.g cytosol containing equal amounts of GFP proteins
(adjusted with cytosol from untransfected cells) in 5 ml blocking
buffer for 1 h at room temperature. Bound proteins are detected by
incubation with anti-GFP antibody, followed by HRP-conjugated
secondary antibody.
In Vitro Ceramide Transfer Assay
[0265] Protein-mediated transfer of ceramide between SUVs is
measured as described previously (Olayioye et al., 2005). The
transfer assay mixture contained donor vesicles (2 nmol lipid/ml)
composed of porcine brain lipids (Avanti Polar Lipids),
pyrene-labeled C.sub.16-ceramide, and
2,4,6-trinitrophenyl-phosphatidylethanolamine (TNP-PE) (88.6:0.4:11
mol %), provided by P. Somerharju, and a 10-fold excess of acceptor
vesicles composed of porcine brain lipids. Fluorescence intensity
is recorded at 395 nm (excitation, 345 nm; slit widths, 4 nm)
before and after the addition of 75 .mu.g cytosol from HEK293T
cells transiently expressing the GFP-tagged CERT wild type and
S132A proteins (see above). Fluorescence intensities are normalized
to (i) the maximum intensity obtained after the addition of Triton
X-100 (0.5% final concentration) and (ii) the maximum GFP
fluorescence, to account for different protein expression
levels.
HRP Transport Assay
[0266] HEK293T cells are cotransfected with ss-HRP-Flag plasmid and
empty vector, pEGFP-N-1-PKD1KD, pcDNA3-Flag-CERT wt and
pcDNA3-Flag-CERT-S132A at a ratio of 1:6.5, respectively. 24 h
post-transfection cells are washed with serum-free media and HRP
secretion is quantified after 0, 1, 3 and 6 h by incubation of
clarified cell supernatant with ECL reagent. Measurements are done
with a luminometer (Lucy2, Anthos) at 450 nm.
siRNA Assay
[0267] COST cells are transfected with a vector encoding
ssHRP-Flag, harvested after 8 hrs, replated into triplicate wells
and then transfected with CERT-specific siRNA oligonucleotides
(siCERT#1, SEQ ID NO.7: 5'-ccacaugacuuacucauuatt-3'; siCERT#2, SEQ
ID NO.8: 5'-gaacag-aggaagcauauaatt-3') using Oligofectamine.TM.
reagent (Invitrogen) according to the manufacturers instructions.
Control cells are either mock transfected or transfected with a
lacZ-specific siRNA (SEQ ID NO.9: 5'-gcggcugccggaauuuacctt-3'). 48
h later, cells are washed and fresh medium is added. The amount of
HRP secreted into the supernatant is measured by a chemiluminescent
assay as described above. Finally, cells are lysed, triplicate
lysates are pooled and analyzed by immunoblotting using tubulin-
and transferrin receptor-specific antibodies.
EXAMPLES
Example 1
Intracellular Product Accumulation Indicates Secretory Bottle
Neck
[0268] A fed-batch process is performed using three different CHO
producer cell clones expressing human IgG antibody (Process A, B
and M, respectively, see FIG. 1). Cell samples are taken every
other day and the amount of intracellular antibody is determined by
FACS analysis. In short, cells are fixed using PBS/4% PFA,
permeabilized and stained with FITC-conjugated anti-human kappa
light chain antibody. Within the first four days, the intracellular
IgG content remains at a constant level. However from day 5 to day
9, the level of intracellular product rises constantly, indicating
an accumulation of either misfolded light chain or even the
complete antibody product within the cell. These data represent the
results of three independent production processes with different
producer cell clones and products and they strongly suggest that
the cell transcribes more antibody RNA than proteins secreted into
the medium and thus points to a post-translational bottle neck
which hinders the complete secretion of the produced antibody (FIG.
1).
Example 2
CERT is Detected by a PKD Substrate Antibody
[0269] PKD is a key regulator at the Golgi complex with
PI4KIII.beta. being the only local substrate identified thus far.
To test whether the Golgi complex-localized CERT protein (SEQ ID
NO.11 and 13) may serve as a substrate for PKD, we make use of a
phosphospecific substrate antibody, termed pMOTIF, raised against
consensus motifs phosphorylated by PKD (Doppler et al., 2005).
HEK293T cells are transfected with expression vectors encoding
Flag-tagged CERT (SEQ ID NO.10) and CERT.sub.L(SEQ ID No.12). The
CERT isoforms are immunoprecipitated with Flag-specific antibodies
and analyzed by Western blotting with the pMOTIF antibody (FIG.
4A). is A pMOTIF signal corresponding to the molecular weight of
CERT (SEQ ID NO.11) and, more weakly, to that of CERT.sub.L.(SEQ ID
No.13) is detected. The weaker detection of the phosphorylated
CERT.sub.L isoform may be related to its known behaviour to form
aggregates, which may impact phosphosite accessibility to kinases
(Raga et al., 2000).
[0270] To investigate whether recognition of CERT by the pMOTIF
antibody is dependent upon PKD, we express CERT together with a
kinase dead variant of PKD1 (K621W) in HEK293T cells. This mutant
has been shown to localize to the Golgi complex and suppressed
PI4KIII.beta. phosphorylation in a dominant negative fashion
(Hausser et al., 2005). Coexpression of inactive PKD abolishes
detection of CERT with the pMOTIF antibody, suggesting that the
pMOTIF signal is indeed due to PKD-mediated CERT phosphorylation
(FIG. 4B).
[0271] Lipid transfer proteins are thought to act at membrane
contact sited, which are formed between the ER and TGN (Levine and
Loewen, 2006), where PKD is localized. Immunofluorescence staining
of Flag-tagged CERT in COS 7 cells coexpressed with GFP-tagged PKD1
verify that the two proteins colocalize at the Golgi complex (FIG.
4C). Together, these data confirm that CERT is a PKD substrate at
the Golgi apparatus.
Example 3
PKD Phosphorylates CERT on Serine 132
[0272] To identify pMOTIF recognition sites in CERT, we search for
potential PKD consensus motifs characterized by a leucine,
isoleucine or valine residue in the -5 and arginine in the -3
position relative to a serine or threonine. Two serines at
positions 132 and 272, matching the PKD consensus motif and
conserved across species (FIG. 5A), are exchanged for alanines by
site-directed mutagenesis. These mutants are expressed in HEK293T
cells and tested for recognition by the pMOTIF antibody.
Interestingly, mutation of serine 132 to alanine abrogate detection
of CERT with the pMOTIF antibody and cause an increase in
electrophoretic mobility, indicative of loss of phosphorylation,
while the S272A mutation does not affect the pMOTIF signal (FIG.
5B). This suggests that serine 132 is a PKD phosphorylation site
specifically recognized by the PKD substrate antibody. To confirm
that PKD is capable of directly phosphorylating this serine residue
in CERT, we perform in vitro kinase assays with purified PKD1 and
recombinant CERT GST-fusion proteins produced in E. coli comprising
the first 138 amino acids of the protein. When the truncated wild
type CERT fusion protein is incubated with PKD1 in the presence of
[.gamma.-.sup.32P]-ATP, incorporation of radioactivity is detected
(FIG. 5C). This is significantly impaired in the case of the
CERT-S132A fusion protein. In vitro PKD phosphorylation of wild
type but not CERT-S132A (SEQ ID NO.15) is further shown to generate
a recognition site for the pMOTIF antibody (FIG. 5D). Taken
together, these results prove that CERT is a genuine PKD substrate
in vitro and in vivo and identify serine 132 as a specific PKD
phosphorylation site in CERT.
Example 4
CERT Phosphorylation on Serine 132 Modulates PI(4)P Binding and
Ceramide Transfer Activity
[0273] Serine 132 is in very close proximity to the CERT PH domain
(amino acids 23-117), making it possible that phosphorylation on
this site affects PI(4)P binding by increasing the local negative
charge. We therefore quantify PI(4)P binding of wild type CERT and
the CERT-S132A mutant (SEQ ID NO.15) by performing protein-lipid
overlay assays. Here, cytosol from HEK293T cells transiently
expressing the CERT variants is incubated with membranes spotted
with a concentration gradient of the different phosphoinositides
and bound CERT proteins are detected via their GFP tag. As reported
previously, the full-length wild type protein demonstrate weak
binding to several phospholipid species, but displays strong
interaction with PI(4)P (Hanada et al., 2003; Levine and Munro,
2002). CERT-S132A binding to PI(4)P is detectable at two- to
fourfold lower concentrations as compared to that of the wild type
protein, suggesting increased affinity of the CERT-S132A mutant to
this phospholipid (FIG. 6A).
[0274] Together, these data imply that CERT, once bound to the
Golgi complex, is phosphorylated by is PKD. This then decreases the
affinity of CERT to PI(4)P and thereby regulates the interaction of
CERT with Golgi membranes.
[0275] As CERT has been shown to function as a lipid transfer
protein (Hanada et al., 2003). We investigate whether CERT
phosphorylation on serine 132 influenced its ability to bind and
transfer ceramide between membranes. To this end, GFP-tagged
versions of wild type CERT (SEQ ID NO.10) and CERT-S132A (SEQ ID
NO.14) are transiently expressed in HEK239T cells and the cytosol
fraction is analyzed for ceramide-specific lipid transfer activity
using a FRET-based assay (FIG. 6B). In this assay, small
unilamellar vesicles containing pyrene-labeled ceramide as a
fluorescent donor and quenching amounts of head group-labeled
TNP-PE are employed (Olayioye et al., 2005; Somerharju, 2002). When
these donor liposomes are mixed with an excess of unlabeled
acceptor liposomes, the increase in pyrene fluorescence is
negligible, indicating minimal spontaneous ceramide transfer to
acceptor membranes (data not shown). Upon addition of wild type
CERT-containing cytosol, a steady increase in fluorescence is
noted, which is not observed when control cytosol of
vector-transfected cells is used (FIG. 6B). Compared to the wild
type protein, CERT-S132A (SEQ ID No.15) displays a higher rate of
lipid transfer, evident from a more rapid increase in pyrene
fluorescence. This suggests that CERT phosphorylation on serine 132
downregulates ceramide transfer activity by decreasing association
of the protein with membranes.
[0276] Previous data have already shown that PKD regulates the
level of PI(4)P at the Golgi complex by phosphorylation-mediated
activation of PI4KIII.beta. (Hausser et al., 2005). Interestingly,
PI4KIII.beta. is critical for the transport of ceramide between the
ER and the Golgi complex (Toth et al., 2006). Accordingly, together
with the data presented here, a dual role for PKD in maintaining
lipid homeostasis of Golgi membranes becomes apparent by
controlling the on-rate is (via PI(4)P levels) and the off-rate
(via direct phosphorylation) of CERT.
Example 5
CERT Regulates PKD Activation and Secretory Transport
[0277] We hypothesize that overexpression of CERT by transferring
ceramide should result in elevated DAG levels and might
consequently stimulate PKD activity. To test this, Flag-tagged CERT
wild type (SEQ ID NO.10) and CERT-S132A (SEQ ID NO.14) are
transiently expressed in HEK293T cells. Whole cells lysates are
prepared 24 h post transfection and subjected to SDSPAGE. PKD
activation is analyzed by immunoblotting with phosphospecific pS916
PKD antibody (FIG. 7A, top panel). Equal loading is verified by
reprobing with PKD-specific antibody (FIG. 7A middle panel).
Expression of CERT proteins is verified by immunoblotting with
Flag-specific antibody (FIG. 7A bottom panel). Compared to the
control, expression of both CERT wild type and CERT-S132A increased
PKD activity, as revealed by analyses with a phosphospecific PKD
antibody. This shows that PKD activation is regulated by CERT
proteins, likely due to increased ceramide delivery and enforced
SM/DAG synthesis.
[0278] To address the question of whether CERT-mediated PKD
activation indeed translates into enhanced secretory transport, we
make use of a plasmid encoding secreted horseradish peroxidase
(HRP-ss) which can be used as reporter for constitutive protein
secretion. HEK293T cells are cotransfected with an expression
plasmid encoding Flag-ss-HRP or empty vector, and PKD1-GFP kinase
dead (KD), Flag-CERT wild type (WT), and Flag-CERT-S132A,
respectively. 24 h post-transfection, cells are washed and fresh
medium is added. The supernatant is analyzed for peroxidase
activity after 0, 1, 3, and 6 h by chemiluminescence. In control
cells, secretion of ss-HRP could be detected within 1 hour and
increased over time (FIG. 7B). Coexpression of kinase dead PKD1,
which inhibits secretory transport of cargo protein almost entirely
abrogates the secretion of ss-HRP into the supernatant. This
confirms that HRP is secreted in a PKD-dependent manner in this
assay. In Contrast, coexpression of CERT is wild type and
CERT-S132A strongly augmented the amount of secreted HRP (FIG. 7B),
the mutant showing even slightly higher values than wild type CERT.
This experiment demonstrates that CERT overexpression stimulates
PKD phosphorylation and in a functional assay enhances secretion of
an extracellular protein into the culture medium by around
2-fold.
[0279] We furthermore investigates whether overexpression of the
CERT-S132A mutant affected its localization and/or caused
morphological changes of the Golgi apparatus. CERT has been
demonstrated to colocalize with the cis/medial-Golgi marker GS28
(Hanada et al., 2003). Immunofluorescence analysis of GFP-tagged
CERT expressed in COST cells shows that the protein localized to
GS28-positive Golgi regions (FIG. 7C). By contrast, in addition to
the partial colocalization with GS28 at the Golgi complex, the
CERT-S132A mutant protein displays a dispersed, punctate staining.
Of note, some of these vesicular structures are found to contain
the cargo protein ss-HRP, providing evidence that these structures
indeed represent Golgi-derived transport carriers (FIG. 7D). This
finding is in accordance with the observed changes in Golgi
membrane structure due to local increases in ceramide levels
(Fukunaga et al., 2000; Weigert et al., 1999).
Example 6
CERT Downregulation by RNA Interference Inhibits Secretory
Transport
[0280] The data presented so far in the present invention clearly
demonstrated that overexpression of CERT enhances protein
secretion. To investigate whether also the opposite is true,
meaning that reduced CERT expression would result in diminished
secretion, siRNA experiments are performed. COST cells are
transfected with a vector encoding ssHRP-Flag, harvested after 8
hrs, replated into triplicate wells and then transfected with
CERT-specific siRNA oligonucleotides (SEQ ID NO.7 and 8) or either
mock or lacZ-specific siRNA (SEQ ID NO.9) as controls. 48 h later,
cells are washed, covered with fresh medium and the amount of HRP
secreted into the is supernatant is measured after the indicated
times.
[0281] As shown in FIG. 8A, activity of HRP is detected after 3
hours and showed equal comparable levels in both control cells. In
contrast, a dramatic reduction of HRP activity is measured in cells
that had been treated with any of the CERT-specific siRNA
oligonucleotides. This indicates that reduced CERT levels lead to
reduced HRP secretion from the cells and further underscores the
important role of CERT in the secretory transport.
[0282] Interestingly, not only protein secretion, but also the
abundance of the transmembrane protein transferrin receptor is
affected by the reduction of CERT (FIG. 8B). When the cells from
FIG. 8A are pooled and the lysates probed with transferrin
receptor-specific antibodies in Western blot experiments, a strong
decrease in the amount of transferrin receptor became apparent,
whereas similar transferrin receptor levels are detected in both
control cells.
[0283] This finding suggests, that the lipid transfer protein CERT
is not only implicated in the transport of secreted but also of
membrane-standing cell-surface proteins. This might not be
surprising as both types of proteins are equally transported in
lipid vesicles from the ER via the Golgi to the plasma membrane and
thus use the same cellular export routes which--as we demonstrate
in the present invention for the first time--are influenced by
CERT.
Example 7
Overexpression of CERT Increases Biopharmaceutical Protein
Production of an Antibody
[0284] (a) An antibody producing CHO cell line (CHO DG44) secreting
humanised anti-CD44v6 IgG antibody BIWA 4 is transfected with an
empty vector (MOCK control) or expression constructs encoding wild
type CERT (SEQ ID NO.10 and 12) or a mutant of CERT bearing the
point-mutation Ser132A (SEQ ID NO.14) and subsequently subjected to
selection to obtain stable cell pools. During six subsequent
passages, supernatant is taken from seed-stock cultures of all
stable cell pools, the IgG titer is determined by ELISA and divided
by the mean number of cells to is calculate the specific
productivity (FIG. 10A). The highest values are seen in the cell
pools harbouring the CERT mutant (SEQ ID No.14), followed by wild
type CERT (SEQ ID No.10 or 12). In both, IgG expression is markedly
enhanced compared to MOCK or untransfected cells. Very similar
results can be obtained if the stable transfectants are subjected
to batch or fed-batch fermentations (FIG. 10B). In each of these
settings, overexpression of both wild type and mutant CERT leads to
increased antibody secretion, indicating that CERT is able to
enhance the specific production capacity of the cells grown in
serial cultures or in bioreactor batch or fed batch cultures.
[0285] b) CHO host cells (CHO DG44) are first transfected with
vectors encoding wild type CERT (SEQ ID NO.10 or 12) or a mutant of
CERT bearing the point-mutation Ser132A (SEQ ID NO.14). Cells are
subjected to selection pressure and cell lines are picked that
demonstrate heterologous expression of CERT or the CERT mutant.
Subsequently these cell lines and in parallel CHO DG 44 wild type
cells are transfected with vectors encoding humanized anti-CD44v6
IgG antibody BIWA 4 as the gene of interest. After a second round
of selection, supernatant is taken from seed-stock cultures of all
stable cell pools over a period of six subsequent passages, the IgG
titer is determined by ELISA and divided by the mean number of
cells to calculate the specific productivity. The highest values
are seen in the cell pools harbouring the CERT mutant (SEQ ID
No.14), followed by wild type CERT (SEQ ID NO.10 or 12). In both,
IgG expression is markedly enhanced compared to cells that don't
have heterologous expression of CERT or CERT mutant. Very similar
results can be obtained if the stable transfectants are subjected
to batch or fed-batch fermentations. In each of these settings,
overexpression of both wild type and mutant CERT leads to increased
antibody secretion, indicating that CERT is able to enhance the
specific production capacity of the cells grown in serial cultures
or in bioreactor batch or fed batch cultures.
[0286] This indicates, that heterologous expression of CERT, and
especially mutant CERT, can enhance antibody secretion in
transiently as well as stably transfected CHO cell lines.
Example 8
Overexpression of CERT Increases Biopharmaceutical Protein
Production of Monocyte Chemoattractant Protein 1 (MCP-1)
[0287] (a) A CHO cell line (CHO DG44) secreting monocyte
chemoattractant protein 1 (MCP-1) is transfected with an empty
vector (MOCK control) or expression constructs encoding wild type
CERT (SEQ ID NO.10 and 12) or a mutant of CERT bearing the
point-mutation Ser132A (SEQ ID NO.14) and subsequently subjected to
selection to obtain stable cell pools. During six subsequent
passages, supernatant is taken from seed-stock cultures of all
stable cell pools, the MCP-1 titer is determined by ELISA and
divided by the mean number of cells to calculate the specific
productivity. The highest values are seen in the cell pools
harbouring the CERT mutant, followed by wild type CERT. In both,
IgG expression is markedly enhanced compared to MOCK or
untransfected cells. Very similar results can be obtained if the
stable transfectants are subjected to batch or fed-batch
fermentations. In each of these settings, overexpression of both
wild type and mutant CERT leads to increased MCP-1 secretion,
indicating that CERT is able to enhance the specific production
capacity of the cells grown in serial cultures or in bioreactor
batch or fed batch cultures.
[0288] b) CHO host cells (CHO DG44) are first transfected with
vectors encoding wild type CERT (SEQ ID NO.10 or 12) or a mutant of
CERT bearing the point-mutation Ser132A (SEQ ID NO.14). Cells are
subjected to selection pressure and cell lines are picked that
demonstrate heterologous expression of CERT or the CERT mutant.
Subsequently these cell lines and in parallel CHO DG 44 wild type
cells are transfected with a vector encoding monocyte
chemoattractant protein 1 (MCP-1) as the gene of interest. After a
second round of selection, supernatant is taken from seed-stock
cultures of all stable cell pools over a period of six subsequent
passages, the MCP-1 titer is determined by ELISA and divided by the
mean number of cells to calculate the specific productivity. The
highest values are seen in the cell pools harbouring the CERT
mutant, followed by wild type CERT. In both, MCP-1 expression is is
markedly enhanced compared to cells that don't have heterologous
expression fo CERT or CERT mutant. Very similar results can be
obtained if the stable transfectants are subjected to batch or
fed-batch fermentations. In each of these settings, overexpression
of both wild type and mutant CERT leads to increased antibody
secretion, indicating that CERT is able to enhance the specific
production capacity of the cells grown in serial cultures or in
bioreactor batch or fed batch cultures.
[0289] This indicates, that heterologous expression of CERT, and
especially mutant CERT, can enhance the secretion of single cell
proteins in transiently as well as stably transfected CHO cell
lines.
Example 9
Overexpression of CERT Increases Biopharmaceutical Protein
Production of Transmembrane Protein Epithelial Growth Factor
Receptor (EGFR)
[0290] (a) A CHO cell line (CHO DG44 expressing transmembrane
protein epithelial growth factor receptor (EGFR) is transfected
with an empty vector (MOCK control) or expression constructs
encoding wild type CERT (SEQ ID NO.10 and 12) or a mutant of CERT
bearing the point-mutation Ser132A (SEQ ID NO.14) and subsequently
subjected to selection to obtain stable cell pools. During six
subsequent passages, cells are taken from seed-stock cultures of
all stable cell pools and the expression level of EGFR is
determined by FACS or Western blotting. The highest values are seen
in the cell pools harbouring the CERT mutant, followed by wild type
CERT. In both, EGFR expression is markedly enhanced compared to
MOCK or untransfected cells. Very similar results can be obtained
if the stable transfectants are subjected to batch or fed-batch
fermentations. In each of these settings, overexpression of both
wild type and mutant CERT leads to increased EGFR expression,
indicating that CERT is able to enhance the specific is production
capacity of the cells grown in serial cultures or in bioreactor
batch or fed batch cultures.
[0291] b) CHO host cells (CHO DG44) are first transfected with
vectors encoding wild type CERT (SEQ ID NO.10 or 12) or a mutant of
CERT bearing the point-mutation Ser132A (SEQ ID NO.14). Cells are
subjected to selection pressure and cell lines are picked that
demonstrate heterologous expression of CERT or the CERT mutant.
Subsequently these cell lines and in parallel CHO DG 44 wild type
cells are transfected with a vector encoding EGFR as the gene of
interest. After a second round of selection, cells are taken from
seed-stock cultures of all stable cell pools for six consecutive
passages and the expression level of EGFR is determined by FACS or
Western blotting. The highest values are seen in the cell pools
harbouring the CERT mutant, followed by wild type CERT. In both,
EGFR expression is markedly enhanced compared to cells that don't
have heterologous expression fo CERT or CERT mutant. Very similar
results can be obtained if the stable transfectants are subjected
to batch or fed-batch fermentations. In each of these settings,
overexpression of both wild type and mutant CERT leads to increased
EGFR expression, indicating that CERT is able to enhance the
specific production capacity of the cells grown in serial cultures
or in bioreactor batch or fed batch cultures.
[0292] This indicates, that heterologous expression of CERT, and
especially mutant CERT, can enhance expression of surface receptors
in transiently as well as stably transfected CHO cell lines.
Example 10
Overexpression of STARD4 Increases Biopharmaceutical Protein
Production of an Antibody
[0293] (a) An antibody producing CHO cell line (CHO DG44) secreting
humanised anti-CD44v6 IgG antibody BIWA 4 is transfected with an
empty vector (MOCK control) or expression constructs encoding
StarD4 (SEQ ID NO.20) and subsequently subjected to selection to
obtain stable cell pools. During six subsequent passages,
supernatant is taken from seed-stock cultures of all stable cell
pools, the IgG titer is determined by ELISA and divided by the mean
number of cells to is calculate the specific productivity. The
highest values are seen in the cell pools harbouring StarD4. IgG
expression is markedly enhanced compared to MOCK or untransfected
cells. Very similar results can be obtained if the stable
transfectants are subjected to batch or fed-batch fermentations. In
each of these settings, overexpression of StarD4 is able to enhance
the specific production capacity of the cells grown in serial
cultures or in bioreactor batch or fed batch cultures.
[0294] b) CHO host cells (CHO DG44) are first transfected with
vectors encoding StarD4. Cells are subjected to selection pressure
and cell lines are picked that demonstrate heterologous expression
of StarD4. Subsequently these cell lines and in parallel CHO DG 44
wild type cells are transfected with vectors encoding humanized
anti-CD44v6 IgG antibody BIWA 4 as the gene of interest. After a
second round of selection, supernatant is taken from seed-stock
cultures of all stable cell pools over a period of six subsequent
passages, the IgG titer is determined by ELISA and divided by the
mean number of cells to calculate the specific productivity. The
highest values are seen in the cell pools harbouring StarD4. IgG
expression is markedly enhanced compared to cells that don't have
heterologous expression of StarD4. Very similar results can be
obtained if the stable transfectants are subjected to batch or
fed-batch fermentations. In each of these settings, overexpression
of StarD4 is able to enhance the specific production capacity of
the cells grown in serial cultures or in bioreactor batch or fed
batch cultures.
[0295] This indicates, that heterologous expression of StarD4, can
enhance antibody secretion in transiently as well as stably
transfected CHO cell lines.
Example 11
Overexpression of CERT Increases Biopharmaceutical Protein
Production of Human Serum Albumin (HSA)
[0296] (a) A CHO cell line (CHO DG44) secreting the single chain
protein HSA is transfected with an empty vector (Mock control) or
expression constructs encoding wild type CERT (SEQ ID NO.10 and 12)
or a mutant of CERT bearing the point-mutation Ser132A (SEQ ID
NO.14) and subsequently subjected to selection to obtain stable
cell pools. During 4 subsequent passages, supernatant is taken from
seed-stock cultures of all stable cell pools, the HSA titer is
determined by ELISA and divided by the mean number of cells to
calculate the specific productivity (FIG. 11A).
[0297] Both, HSA titers and the specific productivity of the HSA
producing cells is significantly enhanced by heterologous
expression of both CERT variants compared to the Mock transfected
control. The highest values are seen in the cell pools harbouring
the CERT mutant, which leads to an increase in the specific
productivity of 51% and an increase in HSA titer of 46% above the
control, followed by wild type CERT, which increases the specific
productivity by 49%.
[0298] Very similar results can be obtained if the stable
transfectants are subjected to batch or fed-batch fermentations
(FIG. 11B). In each of these settings, overexpression of both wild
type and mutant CERT leads to increased HSA secretion, indicating
that CERT is able to enhance the specific production capacity of
the cells grown in serial cultures or under industrial production
conditions such as in bioreactor batch or fed batch cultures.
[0299] (b) and (c) CHO host cells (CHO DG44) are first transfected
with vectors encoding wild type CERT (SEQ ID NO.10 or 12) or a
mutant of CERT bearing the point-mutation Ser132A (SEQ ID NO.14).
Cells are subjected to selection pressure and cell lines are picked
that demonstrate heterologous expression of CERT or the CERT
mutant. Subsequently these cell lines and in parallel CHO DG 44
wild type cells are transfected with a vector encoding human serum
albumin as the gene of interest. After a second round of selection,
supernatant is taken from seed-stock cultures of all stable cell
pools over a period of six subsequent passages, the HSA titer is
determined by ELISA (FIG. 11 C) and divided by the mean number of
cells to calculate the specific productivity (FIG. 11B).
[0300] The highest values are seen in the cell pools harbouring the
CERT mutant, followed by wild type CERT. In both, HSA expression is
markedly enhanced compared to cells that don't have is heterologous
expression fo CERT or CERT mutant. Very similar results can be
obtained if the stable transfectants are subjected to batch or
fed-batch fermentations. In each of these settings, overexpression
of both wild type and mutant CERT leads to increased antibody
secretion, indicating that CERT is able to enhance the specific
production capacity of the cells grown in serial cultures or in
bioreactor batch or fed batch cultures.
[0301] This indicates, that heterologous expression of CERT, and
especially mutant CERT, can enhance the secretion of single-chain
proteins in transiently as well as stably transfected CHO cell
lines.
REFERENCES
[0302] 1. al-Rubeai, M. and Singh, R. P. (1998). Apoptosis in cell
culture. Curr. Opin. Biotechnol. 9, 152-156. [0303] 2. Alpy, F. and
Tomasetto, C. (2005). Give lipids a START: the StAR-related lipid
transfer (START) domain in mammals. J. Cell Sci. 118, 2791-2801.
[0304] 3. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and
Lipman, D. J. (1990). Basic local alignment search tool. J. Mol.
Biol. 215, 403-410. [0305] 4. Ausubel, F. M., Brent, R., Kingston,
R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K.
(2002). Current Protocols in Molecular Biology. John Wiley &
Sons, Inc.). [0306] 5. az Anel, A. M. and Malhotra, V. (2005).
PKCeta is required for beta1gamma2/beta3gamma2- and PKD-mediated
transport to the cell surface and the organization of the Golgi
apparatus. J. Cell Biol. 169, 83-91. [0307] 6. Bard, F., Casano,
L., Mallabiabarrena, A., Wallace, E., Saito, K., Kitayama, H.,
Guizzunti, G., Hu, Y., Wendler, F., Dasgupta, R., Perrimon, N., and
Malhotra, V. (2006). Functional genomics reveals genes involved in
protein secretion and Golgi organization. Nature 439, 604-607.
[0308] 7. Barnes, L. M., Bentley, C. M., Moy, N., and Dickson, A.
J. (2007). Molecular analysis of successful cell line selection in
transfected GS-NS0 myeloma cells. Biotechnol. Bioeng. 96, 337-348.
[0309] 8. Barnes, L. M. and Dickson, A. J. (2006). Mammalian cell
factories for efficient and stable protein expression. Curr. Opin.
Biotechnol. 17, 381-386. [0310] 9. Baron, C. L. and Malhotra, V.
(2002). Role of diacylglycerol in PKD recruitment to the TGN and
protein transport to the plasma membrane. Science 295, 325-328.
[0311] 10. Blobel, C. P. (2005). ADAMs: key components in EGFR
signalling and development. Nat Rev Mol Cell Biol 6, 32-43. [0312]
11. Borth, N., Mattanovich, D., Kunert, R., and Katinger, H.
(2005). Effect of increased expression of protein disulfide
isomerase and heavy chain binding protein on antibody secretion in
a recombinant CHO cell line. Biotechnol. Prog. 21, 106-111. [0313]
12. Brewer, J. W. and Hendershot, L. M. (2005). Building an
antibody factory: a job for the unfolded protein response. Nat
Immunol 6, 23-29. [0314] 13. Chen-Kiang, S. (2003). Cell-cycle
control of plasma cell differentiation and tumorigenesis. Immunol.
Rev. 194, 39-47. [0315] 14. Chiang, G. G. and Sisk, W. P. (2005).
Bcl-x(L) mediates increased production of humanized monoclonal
antibodies in Chinese hamster ovary cells. Biotechnol. Bioeng. 91,
779-792. [0316] 15. Davis, R., Schooley, K., Rasmussen, B., Thomas,
J., and Reddy, P. (2000). Effect of PDI overexpression on
recombinant protein secretion in CHO cells. Biotechnol. Prog. 16,
736-743. [0317] 16. Doppler, H., Storz, P., Li, J., Comb, M. J.,
and Toker, A. (2005). A phosphorylation state-specific antibody
recognizes Hsp27, a novel substrate of protein kinase D. J. Biol.
Chem. 280, 15013-15019. [0318] 17. Dorner, A. J. and Kaufman, R. J.
(1994). The levels of endoplasmic reticulum proteins and is ATP
affect folding and secretion of selective proteins. Biologicals 22,
103-112. [0319] 18. Egeblad, M. and Werb, Z. (2002). New functions
for the matrix metalloproteinases in cancer progression. Nat Rev
Cancer 2, 161-174. [0320] 19. Fukunaga, T., Nagahama, M.,
Hatsuzawa, K., Tani, K., Yamamoto, A., and Tagaya, M. (2000).
Implication of sphingolipid metabolism in the stability of the
Golgi apparatus. J. Cell Sci. 113 (Pt 18), 3299-3307. [0321] 20.
Hanada, K. (2006). Discovery of the molecular machinery CERT for
endoplasmic reticulum-to-Golgi trafficking of ceramide. Mol. Cell
Biochem. 286, 23-31. [0322] 21. Hanada, K., Kumagai, K., Yasuda,
S., Miura, Y., Kawano, M., Fukasawa, M., and Nishijima, M. (2003).
Molecular machinery for non-vesicular trafficking of ceramide.
Nature 426, 803-809. [0323] 22. Hanahan, D. and Weinberg, R. A.
(2000). The hallmarks of cancer. Cell 100, 57-70. [0324] 23. Harris
and Angal (1995). Protein Purification Methods. IRL Press). [0325]
24. Hausser, A., Link, G., Bamberg, L., Burzlaff, A., Lutz, S.,
Pfizenmaier, K., and Johannes, F. J. (2002). Structural
requirements for localization and activation of protein kinase C mu
(PKC mu) at the Golgi compartment. J. Cell Biol 156, 65-74. [0326]
25. Hausser, A., Storz, P., Martens, S., Link, G., Toker, A., and
Pfizenmaier, K. (2005). Protein kinase D regulates vesicular
transport by phosphorylating and activating phosphatidylinositol-4
kinase Illbeta at the Golgi complex. Nat. Cell Biol. 7, 880-886.
[0327] 26. Hooker, A. D., Green, N. H., Baines, A. J., Bull, A. T.,
Jenkins, N., Strange, P. G., and James, D. C. (1999). Constraints
on the transport and glycosylation of recombinant IFN-gamma in
Chinese hamster ovary and insect cells. Biotechnol. Bioeng. 63,
559-572. [0328] 27. Hu, S., Shively, L., Raubitschek, A., Sherman,
M., Williams, L. E., Wong, J. Y., Shively, J. E., and Wu, A. M.
(1996). Minibody: A novel engineered anti-carcinoembryonic antigen
antibody fragment (single-chain Fv-CH3) which exhibits rapid,
high-level targeting of xenografts. Cancer Res. 56, 3055-3061.
[0329] 28. Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M.
S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E.,
Haber, E., Crea, R., and. (1988). Protein engineering of antibody
binding sites: recovery of specific activity in an anti-digoxin
single-chain Fv analogue produced in Escherichia coli. Proc. Natl.
Acad. Sci. U. S. A 85, 5879-5883. [0330] 29. Iglesias, T.,
Cabrera-Poch, N., Mitchell, M. P., Naven, T. J., Rozengurt, E., and
Schiavo, G. (2000). Identification and cloning of Kidins220, a
novel neuronal substrate of protein kinase D. J. Biol. Chem. 275,
40048-40056. [0331] 30. Iwakoshi, N, N., Lee, A. H., and Glimcher,
L. H. (2003). The X-box binding protein-1 transcription factor is
required for plasma cell differentiation and the unfolded protein
response. Immunol Rev 194, 29-38. [0332] 31. Jaggi, M., Rao, P. S.,
Smith, D. J., Wheelock, M. J., Johnson, K. R., Hemstreet, G. P.,
and Balaji, K. C. (2005). E-cadherin phosphorylation by protein
kinase D1/protein kinase C {mu} is associated with altered cellular
aggregation and motility in prostate cancer. Cancer Res. 65,
483-492. [0333] 32. Kaufmann and Fussenegger (2003). Gene Transfer
and Expression in Mammalian Cells. [0334] 33. Kawano, M., Kumagai,
K., Nishijima, M., and Hanada, K. (2006). Efficient trafficking of
ceramide from the endoplasmic reticulum to the Golgi apparatus
requires a VAMP-associated protein-interacting FFAT motif of CERT.
J. Biol Chem. 281, 30279-30288. [0335] 34. Kortt, A. A., Lah, M.,
Oddie, G. W., Gruen, C. L., Burns, J. E., Pearce, L. A., Atwell, J.
L., McCoy, A. J., Howlett, G. J., Metzger, D. W., Webster, R. G.,
and Hudson, P. J. (1997). Single-chain Fv fragments of
anti-neuraminidase antibody NC 10 containing five- and ten-residue
linkers form dimers and with zero-residue linker a trimer. Protein
Eng 10, 423-433. [0336] 35. Levine, T. and Loewen, C. (2006).
Inter-organelle membrane contact sites: through a glass, darkly.
Curr. Opin. Cell Biol 18, 371-378. [0337] 36. Levine, T. P. and
Munro, S. (2002). Targeting of Golgi-specific pleckstrin homology
domains involves both PtdIns 4-kinase-dependent and -independent
components. Curr. Biol. 12, 695-704. [0338] 37. Liljedahl, M.,
Maeda, Y., Colanzi, A., Ayala, I., Van, L. J., and Malhotra, V.
(2001). Protein kinase D regulates the fission of cell surface
destined transport carriers from the trans-Golgi network. Cell 104,
409-420. [0339] 38. Litvak, V., Dahan, N., Ramachandran, S.,
Sabanay, H., and Lev, S. (2005). Maintenance of the diacylglycerol
level in the Golgi apparatus by the Nir2 protein is critical for
Golgi secretory function. Nat. Cell Biol. 7, 225-234. [0340] 39.
Loewen, C. J., Roy, A., and Levine, T. P. (2003). A conserved ER
targeting motif in three families of lipid binding proteins and in
Opi1p binds VAP. EMBO J. 22, 2025-2035. [0341] 40. Lottspeich and
Zorbas (1998). Buch? [0342] 41. Lovejoy, B., Choe, S., Cascio, D.,
McRorie, D. K., DeGrado, W. F., and Eisenberg, D. (1993). Crystal
structure of a synthetic triple-stranded alpha-helical bundle.
Science 259, 1288-1293. [0343] 42. Madden, T. L., Tatusov, R. L.,
and Zhang, J. (1996). Applications of network BLAST server. Methods
Enzymol. 266, 131-141. [0344] 43. Maeda, Y., Beznoussenko, G. V.,
Van, L. J., Mironov, A. A., and Malhotra, V. (2001). Recruitment of
protein kinase D to the trans-Golgi network via the first
cysteine-rich domain. EMBO J. 20, 5982-5990. [0345] 44. Olayioye,
M. A., Vehring, S., Muller, P., Herrmann, A., Schiller, J., Thiele,
C., Lindeman, G. J., Visvader, J. E., and Pomorski, T. (2005).
StarD10, a START domain protein overexpressed in breast cancer,
functions as a phospholipid transfer protein. J. Biol. Chem. 280,
27436-27442. [0346] 45. Overall, C. M. and Kleifeld, O. (2006).
Tumour microenvironment--opinion: validating matrix
metalloproteinases as drug targets and anti-targets for cancer
therapy. Nat Rev Cancer 6, 227-239. [0347] 46. Pack, P., Kujau, M.,
Schroeckh, V., Knupfer, U., Wenderoth, R., Riesenberg, D., and
Pluckthun, A. (1993). Improved bivalent miniantibodies, with
identical avidity as whole antibodies, produced by high cell
density fermentation of Escherichia coli. Biotechnology (N. Y.) 11,
1271-1277. [0348] 47. Pack, P., Muller, K., Zahn, R., and
Pluckthun, A. (1995). Tetravalent miniantibodies with high avidity
assembling in Escherichia coli. J. Mol. Biol. 246, 28-34. [0349]
48. Pak, C. O., Hunt, M. N., Bridges, M. W., Sleigh, M. J., and
Gray, P. P. (1996). Super-CHO-A cell line capable of autocrine
growth under fully defined protein-free conditions. Cytotechnology
V22, 139-146. [0350] 49. Perisic, O., Webb, P. A., Holliger, P.,
Winter, G., and Williams, R. L. (1994). Crystal structure of a
diabody, a bivalent antibody fragment. Structure. 2, 1217-1226.
[0351] 50. Perry, R. J. and Ridgway, N. D. (2005). Molecular
mechanisms and regulation of ceramide transport. Biochim. Biophys.
Acta 1734, 220-234. [0352] 51. Raya, A., Revert-Ros, F.,
Martinez-Martinez, P., Navarro, S., Rosello, E., Vieites, B.,
Granero, F., Forteza, J., and Saus, J. (2000). Goodpasture
antigen-binding protein, the kinase that phosphorylates the
goodpasture antigen, is an alternatively spliced variant implicated
in autoimmune pathogenesis. J. Biol. Chem. 275, 40392-40399. [0353]
52. Robert Scopes (1988). Protein Purification. Springer-Verlag).
[0354] 53. Rykx, A., De, K. L., Mikhalap, S., Vantus, T.,
Seufferlein, T., Vandenheede, J. R., and Van, L. J. (2003). Protein
kinase D: a family affair. FEBS Lett. 546, 81-86. [0355] 54.
Sambrook, J., Fritsch, d. F., and Maniatis, T. (1989). Molecular
Cloning: A Laboratory Manual. (Cold Spring Harbor: Cold Spring
Harbor Laboratory Press). [0356] 55. Schroder, M. (2006). The
unfolded protein response. Mol Biotechnol. 34, 279-290. [0357] 56.
Seth, G., Hossler, P., Yee, J. C., and Hu, W. S. (2006).
Engineering cells for cell culture bioprocessing--physiological
fundamentals. Adv. Biochem. Eng Biotechnol. 101, 119-164. [0358]
57. Shaffer, A. L., Shapiro-Shelef, M., Iwakoshi, N, N., Lee, A.
H., Qian, S. B., Zhao, H., Yu, X., Yang, L., Tan, B. K., Rosenwald,
A., Hurt, E. M., Petroulakis, E., Sonenberg, N., Yewdell, J. W.,
Calame, K., Glimcher, L. H., and Staudt, L. M. (2004). XBP1,
downstream of Blimp-1, expands the secretory apparatus and other
organelles, and increases protein synthesis in plasma cell
differentiation. Immunity. 21, 81-93. [0359] 58. Soccio, R. E. and
Breslow, J. L. (2003). StAR-related lipid transfer (START)
proteins: mediators of intracellular lipid metabolism. J. Biol
Chem. 278, 22183-22186. [0360] 59. Somerharju, P. (2002).
Pyrene-labeled lipids as tools in membrane biophysics and cell
biology. Chem. Phys. Lipids 116, 57-74. [0361] 60. Tigges, M. and
Fussenegger, M. (2006). Xbp1-based engineering of secretory
capacity enhances the productivity of Chinese hamster ovary cells.
Metab Eng. [0362] 61. Toth, B., Balla, A., Ma, H., Knight, Z. A.,
Shokat, K. M., and Balla, T. (2006). Phosphatidylinositol 4-kinase
IIIbeta regulates the transport of ceramide between the endoplasmic
reticulum and Golgi. J. Biol. Chem. 281, 36369-36377. [0363] 62.
Tsujishita, Y. and Hurley, J. H. (2000). Structure and lipid
transport mechanism of a StAR-related domain. Nat. Struct. Biol. 7,
408-414. [0364] 63. Urlaub, G., Kas, E., Carothers, A. M., and
Chasin, L. A. (1983). Deletion of the diploid dihydrofolate
reductase locus from cultured mammalian cells. Cell 33, 405-412.
[0365] 64. Vega, R. B., Harrison, B. C., Meadows, E., Roberts, C.
R., Papst, P. J., Olson, E. N., and McKinsey, T. A. (2004). Protein
kinases C and D mediate agonist-dependent cardiac hypertrophy
through nuclear export of histone deacetylase 5. Mol. Cell Biol.
24, 8374-8385. [0366] 65. Wang, Q. J. (2006). PKD at the crossroads
of DAG and PKC signaling. Trends Pharmacol. Sci. 27, 317-323.
[0367] 66. Wang, Y., Waldron, R. T., Dhaka, A., Patel, A., Riley,
M. M., Rozengurt, E., and Colicelli, J. (2002). The RAS effector
RIN1 directly competes with RAF and is regulated by 14-3-3
proteins. Mol. Cell Biol. 22, 916-926. [0368] 67. Weigert, R.,
Silletta, M. G., Spano, S., Turacchio, G., Cericola, C., Colanzi,
A., Senatore, S., Mancini, R., Polishchuk, E. V., Salmona, M.,
Facchiano, F., Burger, K. N., Mironov, A., Luini, A., and Corda, D.
(1999). CtBP/BARS induces fission of Golgi membranes by acylating
lysophosphatidic acid. Nature 402, 429-433. [0369] 68. Werner, R.
G. (2004). Economic aspects of commercial manufacture of
biopharmaceuticals. J. Biotechnol. 113, 171-182. [0370] 69. Wirtz,
K. W. (2006). Phospholipid transfer proteins in perspective. FEBS
Lett. 580, 5436-5441. [0371] 70. Wurm, F. M. (2004). Production of
recombinant protein therapeutics in cultivated mammalian cells.
Nat. Biotechnol. 22, 1393-1398. [0372] 71. Yeaman, C., Ayala, M.
I., Wright, J. R., Bard, F., Bossard, C., Ang, A., Maeda, Y.,
Seufferlein, T., Mellman, I., Nelson, W. J., and Malhotra, V.
(2004). Protein kinase D regulates basolateral membrane protein
exit from trans-Golgi network. Nat. Cell Biol. 6, 106-112. [0373]
72. Zhang, J. and Madden, T. L. (1997). PowerBLAST: a new network
BLAST application for interactive or automated sequence analysis
and annotation. Genome Res. 7, 649-656.
Sequence CWU 1
1
33128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1cgtcgacatg gcgcaatggt gtccctgg 28228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2ccagggacac cattgcgcca tgtcgacg 28335DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3ggttaaacgt gaggacgcct ggcagaagag actgg 35435DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4ccagtctctt ctgccaggcg tcctcacgtt taacc 35533DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5ggtgtccctg gtgtcttgag caagtggcta ctc 33633DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6gagtagccac ttgctcaaga caccagggac acc 33721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7ccacaugacu uacucauuat t 21821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8gaacagagga agcauauaat t 21921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9gcggcugccg gaauuuacct t 21101797DNAHomo sapiens
10atgtcggata atcagagctg gaactcgtcg ggctcggagg aggatccaga gacggagtct
60gggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacat tcatgggtgg
120caggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatc
tgaagatgaa 180acagagtatg gctgcagagg atccatctgt cttagcaagg
ctgtcatcac acctcacgat 240tttgatgaat gtcgatttga tattagtgta
aatgatagtg tttggtatct tcgtgctcag 300gatccagatc atagacagca
atggatagat gccattgaac agcacaagac tgaatctgga 360tatggatctg
aatccagctt gcgtcgacat ggctcaatgg tgtccctggt gtctggagca
420agtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagttt
acgtgagaag 480ttggctgaaa tggaaacatt tagagacatc ttatgtagac
aagttgacac gctacagaag 540tactttgatg cctgtgctga tgctgtctct
aaggatgaac ttcaaaggga taaagtggta 600gaagatgatg aagatgactt
tcctacaacg cgttctgatg gtgacttctt gcatagtacc 660aacggcaata
aagaaaagtt atttccacat gtgacaccaa aaggaattaa tggtatagac
720tttaaagggg aagcgataac ttttaaagca actactgctg gaatccttgc
aacactttct 780cattgtattg aactaatggt taaacgtgag gacagctggc
agaagagact ggataaggaa 840actgagaaga aaagaagaac agaggaagca
tataaaaatg caatgacaga acttaagaaa 900aaatcccact ttggaggacc
agattatgaa gaaggcccta acagtctgat taatgaagaa 960gagttctttg
atgctgttga agctgctctt gacagacaag ataaaataga agaacagtca
1020cagagtgaaa aggtgagatt acattggcct acatccttgc cctctggaga
tgccttttct 1080tctgtgggga cacatagatt tgtccaaaag gttgaagaga
tggtgcagaa ccacatgact 1140tactcattac aggatgtagg cggagatgcc
aattggcagt tggttgtaga agaaggagaa 1200atgaaggtat acagaagaga
agtagaagaa aatgggattg ttctggatcc tttaaaagct 1260acccatgcag
ttaaaggcgt cacaggacat gaagtctgca attatttctg gaatgttgac
1320gttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaac
attagctgat 1380aatgcaatca tcatttatca aacacacaag agggtgtggc
ctgcttctca gcgagacgta 1440ttatatcttt ctgtcattcg aaagatacca
gccttgactg aaaatgaccc tgaaacttgg 1500atagtttgta atttttctgt
ggatcatgac agtgctcctc taaacaaccg atgtgtccgt 1560gccaaaataa
atgttgctat gatttgtcaa accttggtaa gcccaccaga gggaaaccag
1620gaaattagca gggacaacat tctatgcaag attacatatg tagctaatgt
gaaccctgga 1680ggatgggcac cagcctcagt gttaagggca gtggcaaagc
gagagtatcc taaatttcta 1740aaacgtttta cttcttacgt ccaagaaaaa
actgcaggaa agcctatttt gttctag 179711598PRTHomo sapiens 11Met Ser
Asp Asn Gln Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp Pro1 5 10 15Glu
Thr Glu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser Lys 20 25
30Trp Thr Asn Tyr Ile His Gly Trp Gln Asp Arg Trp Val Val Leu Lys
35 40 45Asn Asn Ala Leu Ser Tyr Tyr Lys Ser Glu Asp Glu Thr Glu Tyr
Gly 50 55 60Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala Val Ile Thr Pro
His Asp65 70 75 80Phe Asp Glu Cys Arg Phe Asp Ile Ser Val Asn Asp
Ser Val Trp Tyr 85 90 95Leu Arg Ala Gln Asp Pro Asp His Arg Gln Gln
Trp Ile Asp Ala Ile 100 105 110Glu Gln His Lys Thr Glu Ser Gly Tyr
Gly Ser Glu Ser Ser Leu Arg 115 120 125Arg His Gly Ser Met Val Ser
Leu Val Ser Gly Ala Ser Gly Tyr Ser 130 135 140Ala Thr Ser Thr Ser
Ser Phe Lys Lys Gly His Ser Leu Arg Glu Lys145 150 155 160Leu Ala
Glu Met Glu Thr Phe Arg Asp Ile Leu Cys Arg Gln Val Asp 165 170
175Thr Leu Gln Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys Asp
180 185 190Glu Leu Gln Arg Asp Lys Val Val Glu Asp Asp Glu Asp Asp
Phe Pro 195 200 205Thr Thr Arg Ser Asp Gly Asp Phe Leu His Ser Thr
Asn Gly Asn Lys 210 215 220Glu Lys Leu Phe Pro His Val Thr Pro Lys
Gly Ile Asn Gly Ile Asp225 230 235 240Phe Lys Gly Glu Ala Ile Thr
Phe Lys Ala Thr Thr Ala Gly Ile Leu 245 250 255Ala Thr Leu Ser His
Cys Ile Glu Leu Met Val Lys Arg Glu Asp Ser 260 265 270Trp Gln Lys
Arg Leu Asp Lys Glu Thr Glu Lys Lys Arg Arg Thr Glu 275 280 285Glu
Ala Tyr Lys Asn Ala Met Thr Glu Leu Lys Lys Lys Ser His Phe 290 295
300Gly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Ser Leu Ile Asn Glu
Glu305 310 315 320Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg
Gln Asp Lys Ile 325 330 335Glu Glu Gln Ser Gln Ser Glu Lys Val Arg
Leu His Trp Pro Thr Ser 340 345 350Leu Pro Ser Gly Asp Ala Phe Ser
Ser Val Gly Thr His Arg Phe Val 355 360 365Gln Lys Val Glu Glu Met
Val Gln Asn His Met Thr Tyr Ser Leu Gln 370 375 380Asp Val Gly Gly
Asp Ala Asn Trp Gln Leu Val Val Glu Glu Gly Glu385 390 395 400Met
Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu Asp 405 410
415Pro Leu Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu Val
420 425 430Cys Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp Trp Glu
Thr Thr 435 440 445Ile Glu Asn Phe His Val Val Glu Thr Leu Ala Asp
Asn Ala Ile Ile 450 455 460Ile Tyr Gln Thr His Lys Arg Val Trp Pro
Ala Ser Gln Arg Asp Val465 470 475 480Leu Tyr Leu Ser Val Ile Arg
Lys Ile Pro Ala Leu Thr Glu Asn Asp 485 490 495Pro Glu Thr Trp Ile
Val Cys Asn Phe Ser Val Asp His Asp Ser Ala 500 505 510Pro Leu Asn
Asn Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile 515 520 525Cys
Gln Thr Leu Val Ser Pro Pro Glu Gly Asn Gln Glu Ile Ser Arg 530 535
540Asp Asn Ile Leu Cys Lys Ile Thr Tyr Val Ala Asn Val Asn Pro
Gly545 550 555 560Gly Trp Ala Pro Ala Ser Val Leu Arg Ala Val Ala
Lys Arg Glu Tyr 565 570 575Pro Lys Phe Leu Lys Arg Phe Thr Ser Tyr
Val Gln Glu Lys Thr Ala 580 585 590Gly Lys Pro Ile Leu Phe
595122389DNAHomo sapiens 12gcaggaagat ggcggcggta gcggaggtgt
gagtggacgc gggactcagc ggccggattt 60tctcttccct tcttttccct tttccttccc
tatttgaaat tggcatcgag ggggctaagt 120tcgggtggca gcgccgggcg
caacgcaggg gtcacggcga cggcggcggc ggctgacggc 180tggaagggta
ggcttcattc accgctcgtc ctccttcctc gctccgctcg gtgtcaggcg
240cggcggcggc gcggcgggcg gacttcgtcc ctcctcctgc tcccccccac
accggagcgg 300gcactcttcg cttcgccatc ccccgaccct tcaccccgag
gactgggcgc ctcctccggc 360gcagctgagg gagcgggggc cggtctcctg
ctcggttgtc gagcctccat gtcggataat 420cagagctgga actcgtcggg
ctcggaggag gatccagaga cggagtctgg gccgcctgtg 480gagcgctgcg
gggtcctcag taagtggaca aactacattc atgggtggca ggatcgttgg
540gtagttttga aaaataatgc tctgagttac tacaaatctg aagatgaaac
agagtatggc 600tgcagaggat ccatctgtct tagcaaggct gtcatcacac
ctcacgattt tgatgaatgt 660cgatttgata ttagtgtaaa tgatagtgtt
tggtatcttc gtgctcagga tccagatcat 720agacagcaat ggatagatgc
cattgaacag cacaagactg aatctggata tggatctgaa 780tccagcttgc
gtcgacatgg ctcaatggtg tccctggtgt ctggagcaag tggctactct
840gcaacatcca cctcttcatt caagaaaggc cacagtttac gtgagaagtt
ggctgaaatg 900gaaacattta gagacatctt atgtagacaa gttgacacgc
tacagaagta ctttgatgcc 960tgtgctgatg ctgtctctaa ggatgaactt
caaagggata aagtggtaga agatgatgaa 1020gatgactttc ctacaacgcg
ttctgatggt gacttcttgc atagtaccaa cggcaataaa 1080gaaaagttat
ttccacatgt gacaccaaaa ggaattaatg gtatagactt taaaggggaa
1140gcgataactt ttaaagcaac tactgctgga atccttgcaa cactttctca
ttgtattgaa 1200ctaatggtta aacgtgagga cagctggcag aagagactgg
ataaggaaac tgagaagaaa 1260agaagaacag aggaagcata taaaaatgca
atgacagaac ttaagaaaaa atcccacttt 1320ggaggaccag attatgaaga
aggccctaac agtctgatta atgaagaaga gttctttgat 1380gctgttgaag
ctgctcttga cagacaagat aaaatagaag aacagtcaca gagtgaaaag
1440gtgagattac attggcctac atccttgccc tctggagatg ccttttcttc
tgtggggaca 1500catagatttg tccaaaagcc ctatagtcgc tcttcctcca
tgtcttccat tgatctagtc 1560agtgcctctg atgatgttca cagattcagc
tcccaggttg aagagatggt gcagaaccac 1620atgacttact cattacagga
tgtaggcgga gatgccaatt ggcagttggt tgtagaagaa 1680ggagaaatga
aggtatacag aagagaagta gaagaaaatg ggattgttct ggatccttta
1740aaagctaccc atgcagttaa aggcgtcaca ggacatgaag tctgcaatta
tttctggaat 1800gttgacgttc gcaatgactg ggaaacaact atagaaaact
ttcatgtggt ggaaacatta 1860gctgataatg caatcatcat ttatcaaaca
cacaagaggg tgtggcctgc ttctcagcga 1920gacgtattat atctttctgt
cattcgaaag ataccagcct tgactgaaaa tgaccctgaa 1980acttggatag
tttgtaattt ttctgtggat catgacagtg ctcctctaaa caaccgatgt
2040gtccgtgcca aaataaatgt tgctatgatt tgtcaaacct tggtaagccc
accagaggga 2100aaccaggaaa ttagcaggga caacattcta tgcaagatta
catatgtagc taatgtgaac 2160cctggaggat gggcaccagc ctcagtgtta
agggcagtgg caaagcgaga gtatcctaaa 2220tttctaaaac gttttacttc
ttacgtccaa gaaaaaactg caggaaagcc tattttgttc 2280tagtattaac
aggtactaga agatatgttt tatctttttt taactttatt tgactaatat
2340gactgtcaat actaaaattt agttgttgaa agtatttact atgtttttt
238913624PRTHomo sapiens 13Met Ser Asp Asn Gln Ser Trp Asn Ser Ser
Gly Ser Glu Glu Asp Pro1 5 10 15Glu Thr Glu Ser Gly Pro Pro Val Glu
Arg Cys Gly Val Leu Ser Lys 20 25 30Trp Thr Asn Tyr Ile His Gly Trp
Gln Asp Arg Trp Val Val Leu Lys 35 40 45Asn Asn Ala Leu Ser Tyr Tyr
Lys Ser Glu Asp Glu Thr Glu Tyr Gly 50 55 60Cys Arg Gly Ser Ile Cys
Leu Ser Lys Ala Val Ile Thr Pro His Asp65 70 75 80Phe Asp Glu Cys
Arg Phe Asp Ile Ser Val Asn Asp Ser Val Trp Tyr 85 90 95Leu Arg Ala
Gln Asp Pro Asp His Arg Gln Gln Trp Ile Asp Ala Ile 100 105 110Glu
Gln His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg 115 120
125Arg His Gly Ser Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser
130 135 140Ala Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg
Glu Lys145 150 155 160Leu Ala Glu Met Glu Thr Phe Arg Asp Ile Leu
Cys Arg Gln Val Asp 165 170 175Thr Leu Gln Lys Tyr Phe Asp Ala Cys
Ala Asp Ala Val Ser Lys Asp 180 185 190Glu Leu Gln Arg Asp Lys Val
Val Glu Asp Asp Glu Asp Asp Phe Pro 195 200 205Thr Thr Arg Ser Asp
Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys 210 215 220Glu Lys Leu
Phe Pro His Val Thr Pro Lys Gly Ile Asn Gly Ile Asp225 230 235
240Phe Lys Gly Glu Ala Ile Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu
245 250 255Ala Thr Leu Ser His Cys Ile Glu Leu Met Val Lys Arg Glu
Asp Ser 260 265 270Trp Gln Lys Arg Leu Asp Lys Glu Thr Glu Lys Lys
Arg Arg Thr Glu 275 280 285Glu Ala Tyr Lys Asn Ala Met Thr Glu Leu
Lys Lys Lys Ser His Phe 290 295 300Gly Gly Pro Asp Tyr Glu Glu Gly
Pro Asn Ser Leu Ile Asn Glu Glu305 310 315 320Glu Phe Phe Asp Ala
Val Glu Ala Ala Leu Asp Arg Gln Asp Lys Ile 325 330 335Glu Glu Gln
Ser Gln Ser Glu Lys Val Arg Leu His Trp Pro Thr Ser 340 345 350Leu
Pro Ser Gly Asp Ala Phe Ser Ser Val Gly Thr His Arg Phe Val 355 360
365Gln Lys Pro Tyr Ser Arg Ser Ser Ser Met Ser Ser Ile Asp Leu Val
370 375 380Ser Ala Ser Asp Asp Val His Arg Phe Ser Ser Gln Val Glu
Glu Met385 390 395 400Val Gln Asn His Met Thr Tyr Ser Leu Gln Asp
Val Gly Gly Asp Ala 405 410 415Asn Trp Gln Leu Val Val Glu Glu Gly
Glu Met Lys Val Tyr Arg Arg 420 425 430Glu Val Glu Glu Asn Gly Ile
Val Leu Asp Pro Leu Lys Ala Thr His 435 440 445Ala Val Lys Gly Val
Thr Gly His Glu Val Cys Asn Tyr Phe Trp Asn 450 455 460Val Asp Val
Arg Asn Asp Trp Glu Thr Thr Ile Glu Asn Phe His Val465 470 475
480Val Glu Thr Leu Ala Asp Asn Ala Ile Ile Ile Tyr Gln Thr His Lys
485 490 495Arg Val Trp Pro Ala Ser Gln Arg Asp Val Leu Tyr Leu Ser
Val Ile 500 505 510Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp Pro Glu
Thr Trp Ile Val 515 520 525Cys Asn Phe Ser Val Asp His Asp Ser Ala
Pro Leu Asn Asn Arg Cys 530 535 540Val Arg Ala Lys Ile Asn Val Ala
Met Ile Cys Gln Thr Leu Val Ser545 550 555 560Pro Pro Glu Gly Asn
Gln Glu Ile Ser Arg Asp Asn Ile Leu Cys Lys 565 570 575Ile Thr Tyr
Val Ala Asn Val Asn Pro Gly Gly Trp Ala Pro Ala Ser 580 585 590Val
Leu Arg Ala Val Ala Lys Arg Glu Tyr Pro Lys Phe Leu Lys Arg 595 600
605Phe Thr Ser Tyr Val Gln Glu Lys Thr Ala Gly Lys Pro Ile Leu Phe
610 615 620141797DNAHomo sapiens 14atgtcggata atcagagctg gaactcgtcg
ggctcggagg aggatccaga gacggagtct 60gggccgcctg tggagcgctg cggggtcctc
agtaagtgga caaactacat tcatgggtgg 120caggatcgtt gggtagtttt
gaaaaataat gctctgagtt actacaaatc tgaagatgaa 180acagagtatg
gctgcagagg atccatctgt cttagcaagg ctgtcatcac acctcacgat
240tttgatgaat gtcgatttga tattagtgta aatgatagtg tttggtatct
tcgtgctcag 300gatccagatc atagacagca atggatagat gccattgaac
agcacaagac tgaatctgga 360tatggatctg aatccagctt gcgtcgacat
ggcgcaatgg tgtccctggt gtctggagca 420agtggctact ctgcaacatc
cacctcttca ttcaagaaag gccacagttt acgtgagaag 480ttggctgaaa
tggaaacatt tagagacatc ttatgtagac aagttgacac gctacagaag
540tactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaaggga
taaagtggta 600gaagatgatg aagatgactt tcctacaacg cgttctgatg
gtgacttctt gcatagtacc 660aacggcaata aagaaaagtt atttccacat
gtgacaccaa aaggaattaa tggtatagac 720tttaaagggg aagcgataac
ttttaaagca actactgctg gaatccttgc aacactttct 780cattgtattg
aactaatggt taaacgtgag gacagctggc agaagagact ggataaggaa
840actgagaaga aaagaagaac agaggaagca tataaaaatg caatgacaga
acttaagaaa 900aaatcccact ttggaggacc agattatgaa gaaggcccta
acagtctgat taatgaagaa 960gagttctttg atgctgttga agctgctctt
gacagacaag ataaaataga agaacagtca 1020cagagtgaaa aggtgagatt
acattggcct acatccttgc cctctggaga tgccttttct 1080tctgtgggga
cacatagatt tgtccaaaag gttgaagaga tggtgcagaa ccacatgact
1140tactcattac aggatgtagg cggagatgcc aattggcagt tggttgtaga
agaaggagaa 1200atgaaggtat acagaagaga agtagaagaa aatgggattg
ttctggatcc tttaaaagct 1260acccatgcag ttaaaggcgt cacaggacat
gaagtctgca attatttctg gaatgttgac 1320gttcgcaatg actgggaaac
aactatagaa aactttcatg tggtggaaac attagctgat 1380aatgcaatca
tcatttatca aacacacaag agggtgtggc ctgcttctca gcgagacgta
1440ttatatcttt ctgtcattcg aaagatacca gccttgactg aaaatgaccc
tgaaacttgg 1500atagtttgta atttttctgt ggatcatgac agtgctcctc
taaacaaccg atgtgtccgt 1560gccaaaataa atgttgctat gatttgtcaa
accttggtaa gcccaccaga gggaaaccag 1620gaaattagca gggacaacat
tctatgcaag attacatatg tagctaatgt gaaccctgga 1680ggatgggcac
cagcctcagt gttaagggca gtggcaaagc gagagtatcc taaatttcta
1740aaacgtttta cttcttacgt ccaagaaaaa actgcaggaa agcctatttt gttctag
179715598PRTHomo sapiens 15Met Ser Asp Asn Gln Ser Trp Asn Ser Ser
Gly Ser Glu Glu Asp Pro1 5 10 15Glu Thr Glu Ser Gly Pro Pro Val Glu
Arg Cys Gly Val Leu Ser Lys 20 25 30Trp Thr Asn Tyr Ile His Gly Trp
Gln Asp Arg Trp Val Val Leu Lys 35 40 45Asn Asn Ala Leu Ser Tyr Tyr
Lys
Ser Glu Asp Glu Thr Glu Tyr Gly 50 55 60Cys Arg Gly Ser Ile Cys Leu
Ser Lys Ala Val Ile Thr Pro His Asp65 70 75 80Phe Asp Glu Cys Arg
Phe Asp Ile Ser Val Asn Asp Ser Val Trp Tyr 85 90 95Leu Arg Ala Gln
Asp Pro Asp His Arg Gln Gln Trp Ile Asp Ala Ile 100 105 110Glu Gln
His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg 115 120
125Arg His Gly Ala Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser
130 135 140Ala Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg
Glu Lys145 150 155 160Leu Ala Glu Met Glu Thr Phe Arg Asp Ile Leu
Cys Arg Gln Val Asp 165 170 175Thr Leu Gln Lys Tyr Phe Asp Ala Cys
Ala Asp Ala Val Ser Lys Asp 180 185 190Glu Leu Gln Arg Asp Lys Val
Val Glu Asp Asp Glu Asp Asp Phe Pro 195 200 205Thr Thr Arg Ser Asp
Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys 210 215 220Glu Lys Leu
Phe Pro His Val Thr Pro Lys Gly Ile Asn Gly Ile Asp225 230 235
240Phe Lys Gly Glu Ala Ile Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu
245 250 255Ala Thr Leu Ser His Cys Ile Glu Leu Met Val Lys Arg Glu
Asp Ser 260 265 270Trp Gln Lys Arg Leu Asp Lys Glu Thr Glu Lys Lys
Arg Arg Thr Glu 275 280 285Glu Ala Tyr Lys Asn Ala Met Thr Glu Leu
Lys Lys Lys Ser His Phe 290 295 300Gly Gly Pro Asp Tyr Glu Glu Gly
Pro Asn Ser Leu Ile Asn Glu Glu305 310 315 320Glu Phe Phe Asp Ala
Val Glu Ala Ala Leu Asp Arg Gln Asp Lys Ile 325 330 335Glu Glu Gln
Ser Gln Ser Glu Lys Val Arg Leu His Trp Pro Thr Ser 340 345 350Leu
Pro Ser Gly Asp Ala Phe Ser Ser Val Gly Thr His Arg Phe Val 355 360
365Gln Lys Val Glu Glu Met Val Gln Asn His Met Thr Tyr Ser Leu Gln
370 375 380Asp Val Gly Gly Asp Ala Asn Trp Gln Leu Val Val Glu Glu
Gly Glu385 390 395 400Met Lys Val Tyr Arg Arg Glu Val Glu Glu Asn
Gly Ile Val Leu Asp 405 410 415Pro Leu Lys Ala Thr His Ala Val Lys
Gly Val Thr Gly His Glu Val 420 425 430Cys Asn Tyr Phe Trp Asn Val
Asp Val Arg Asn Asp Trp Glu Thr Thr 435 440 445Ile Glu Asn Phe His
Val Val Glu Thr Leu Ala Asp Asn Ala Ile Ile 450 455 460Ile Tyr Gln
Thr His Lys Arg Val Trp Pro Ala Ser Gln Arg Asp Val465 470 475
480Leu Tyr Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp
485 490 495Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val Asp His Asp
Ser Ala 500 505 510Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn
Val Ala Met Ile 515 520 525Cys Gln Thr Leu Val Ser Pro Pro Glu Gly
Asn Gln Glu Ile Ser Arg 530 535 540Asp Asn Ile Leu Cys Lys Ile Thr
Tyr Val Ala Asn Val Asn Pro Gly545 550 555 560Gly Trp Ala Pro Ala
Ser Val Leu Arg Ala Val Ala Lys Arg Glu Tyr 565 570 575Pro Lys Phe
Leu Lys Arg Phe Thr Ser Tyr Val Gln Glu Lys Thr Ala 580 585 590Gly
Lys Pro Ile Leu Phe 59516669DNAHomo sapiens 16agatttgtcc aaaaggttga
agagatggtg cagaaccaca tgacttactc attacaggat 60gtaggcggag atgccaattg
gcagttggtt gtagaagaag gagaaatgaa ggtatacaga 120agagaagtag
aagaaaatgg gattgttctg gatcctttaa aagctaccca tgcagttaaa
180ggcgtcacag gacatgaagt ctgcaattat ttctggaatg ttgacgttcg
caatgactgg 240gaaacaacta tagaaaactt tcatgtggtg gaaacattag
ctgataatgc aatcatcatt 300tatcaaacac acaagagggt gtggcctgct
tctcagcgag acgtattata tctttctgtc 360attcgaaaga taccagcctt
gactgaaaat gaccctgaaa cttggatagt ttgtaatttt 420tctgtggatc
atgacagtgc tcctctaaac aaccgatgtg tccgtgccaa aataaatgtt
480gctatgattt gtcaaacctt ggtaagccca ccagagggaa accaggaaat
tagcagggac 540aacattctat gcaagattac atatgtagct aatgtgaacc
ctggaggatg ggcaccagcc 600tcagtgttaa gggcagtggc aaagcgagag
tatcctaaat ttctaaaacg ttttacttct 660tacgtccaa 66917223PRTHomo
sapiens 17Arg Phe Val Gln Lys Val Glu Glu Met Val Gln Asn His Met
Thr Tyr1 5 10 15Ser Leu Gln Asp Val Gly Gly Asp Ala Asn Trp Gln Leu
Val Val Glu 20 25 30Glu Gly Glu Met Lys Val Tyr Arg Arg Glu Val Glu
Glu Asn Gly Ile 35 40 45Val Leu Asp Pro Leu Lys Ala Thr His Ala Val
Lys Gly Val Thr Gly 50 55 60His Glu Val Cys Asn Tyr Phe Trp Asn Val
Asp Val Arg Asn Asp Trp65 70 75 80Glu Thr Thr Ile Glu Asn Phe His
Val Val Glu Thr Leu Ala Asp Asn 85 90 95Ala Ile Ile Ile Tyr Gln Thr
His Lys Arg Val Trp Pro Ala Ser Gln 100 105 110Arg Asp Val Leu Tyr
Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr 115 120 125Glu Asn Asp
Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val Asp His 130 135 140Asp
Ser Ala Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn Val145 150
155 160Ala Met Ile Cys Gln Thr Leu Val Ser Pro Pro Glu Gly Asn Gln
Glu 165 170 175Ile Ser Arg Asp Asn Ile Leu Cys Lys Ile Thr Tyr Val
Ala Asn Val 180 185 190Asn Pro Gly Gly Trp Ala Pro Ala Ser Val Leu
Arg Ala Val Ala Lys 195 200 205Arg Glu Tyr Pro Lys Phe Leu Lys Arg
Phe Thr Ser Tyr Val Gln 210 215 22018658DNAHomo sapiens
18caggttgaag agatggtgca gaaccacatg acttactcat tacaggatgt aggcggagat
60gccaattggc agttggttgt agaagaagga gaaatgaagg tatacagaag agaagtagaa
120gaaaatggga ttgttctgga tcctttaaaa gctacccatg cagttaaagg
cgtcacagga 180catgaagtct gcaattattt ctggaatgtt gacgttcgca
atgactggga aacaactata 240gaaaactttc atgtggtgga aacattagct
gataatgcaa tcatcattta tcaaacacac 300aagagggtgt ggcctgcttc
tcagcgagac gtattatatc tttctgtcat tcgaaagata 360ccagccttga
ctgaaaatga ccctgaaact tggatagttt gtaatttttc tgtggatcat
420gacagtgctc ctctaaacaa ccgatgtgtc cgtgccaaaa taaatgttgc
tatgatttgt 480caaaccttgg taagcccacc agagggaaac caggaaatta
gcagggacaa cattctatgc 540aagattacat atgtagctaa tgtgaaccct
ggaggatggg caccagcctc agtgttaagg 600gcagtggcaa agcgagagta
tcctaaattt ctaaaacgtt ttacttctta cgtccaag 65819219PRTHomo sapiens
19Gln Val Glu Glu Met Val Gln Asn His Met Thr Tyr Ser Leu Gln Asp1
5 10 15Val Gly Gly Asp Ala Asn Trp Gln Leu Val Val Glu Glu Gly Glu
Met 20 25 30Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu
Asp Pro 35 40 45Leu Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His
Glu Val Cys 50 55 60Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp Trp
Glu Thr Thr Ile65 70 75 80Glu Asn Phe His Val Val Glu Thr Leu Ala
Asp Asn Ala Ile Ile Ile 85 90 95Tyr Gln Thr His Lys Arg Val Trp Pro
Ala Ser Gln Arg Asp Val Leu 100 105 110Tyr Leu Ser Val Ile Arg Lys
Ile Pro Ala Leu Thr Glu Asn Asp Pro 115 120 125Glu Thr Trp Ile Val
Cys Asn Phe Ser Val Asp His Asp Ser Ala Pro 130 135 140Leu Asn Asn
Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile Cys145 150 155
160Gln Thr Leu Val Ser Pro Pro Glu Gly Asn Gln Glu Ile Ser Arg Asp
165 170 175Asn Ile Leu Cys Lys Ile Thr Tyr Val Ala Asn Val Asn Pro
Gly Gly 180 185 190Trp Ala Pro Ala Ser Val Leu Arg Ala Val Ala Lys
Arg Glu Tyr Pro 195 200 205Lys Phe Leu Lys Arg Phe Thr Ser Tyr Val
Gln 210 215202264DNAHomo sapiens 20actgttgaga gcggtgtgag gtgcttggta
gcgcgccgta gctgcttcca cgtccttgct 60tcacctcagg taaagagaga agtaatggaa
ggcctgtctg atgttgcttc ttttgcaact 120aaacttaaaa acactctcat
ccagtaccat agcattgaag aagataagtg gcgagttgct 180aagaaaacga
aagatgtaac tgtttggaga aaaccctcag aagaatttaa tggatatctc
240tacaaagccc aaggtgttat agatgacctt gtctatagta taatagacca
tatacgccca 300gggccttgtc gtttggattg ggacagcttg atgacttctt
tggatattct ggagaacttt 360gaagagaatt gctgtgtgat gcgttacact
actgctggtc agctttggaa tataatttcc 420ccaagagaat ttgttgattt
ctcctatact gtgggctata aagaagggct tttatcttgt 480ggaataagtc
ttgactggga tgaaaagaga ccagaatttg ttcgaggata taaccatccc
540tgtggttggt tttgtgttcc acttaaagac aacccaaacc agagtctttt
gacaggatat 600attcagacag atctgcgtgg gatgattcct cagtctgcgg
tagatacagc catggcaagc 660actttaacca acttctatgg tgatttacga
aaagctttat gagaggcaaa atacattcaa 720acttgtagta ctacagatca
actctctcag ctacatggcc tgtaaaaatc attgattcca 780cttttctgca
tagccggtag aaaaatttga aatgtttttg gttcactagt acaatgtttg
840gttttattcc taaagtaaat agctatctaa gagagggcat tttcactttt
ttttttttaa 900attttgagac aggctctcac tctgttgccc atgctggagg
gcagtggtat gatcacagct 960cactgcagct ttgatctgac cgctcaaggg
gttattctac ctcagcctcc tgaatagctg 1020ggaatacagg tgcacgccac
tatgcatggc taatttttgt ttaatttttt gtagagatgt 1080ggtcacactg
tgttgcccag gctggtcttg aactcctggc ctcaagtcat tccccacctt
1140agcctcccaa agtgttggga ttataagcgt gagccaccat gcctggcccc
aatttaaaat 1200gtggaattca gttggtgtcc aagacttatc ttgagactct
taaaagcatc agtctgtaac 1260tagaacaaat acagtcttag atttacccaa
gtgcctagat atcattttat aatgattaga 1320attgagtatt gtgggtcccc
taattctgtg ggtgccttaa gtgagaattt ctaaatgatt 1380ttcacattct
aaatgacttt gggttttgaa ctctccatct agtttacttc taaaatggga
1440acttgaggca attcaggtat ccaggcaaat ctttgtatat atttttttgt
gtacatgcac 1500acatctcgaa atccatttcc gtgtttaatg ttagttgttt
atgtgttagt attcctgtgt 1560ctactgtttt gttgttgtta atatgggtaa
agtgagccct gaaatacatg ctaaacaaga 1620catgaaattc agaaaggtac
atagtgtttc aagtgcatgg tagtttgatc tgtgttttac 1680tttattgtgt
tttcttgagt gtaaagaaag aataaatcaa agttcttcat acccattttg
1740acaaagtgga acagtggagc tgttttttgc ttttgttttt atttattttt
tgccactggt 1800gatgatagat ttcaaaaaac aaaaggtggc agcagcacaa
tgttcatggt gaattatctc 1860atagtatcta gattgatcaa gatctgacag
aaggaatgca caaaggattc tatattctta 1920atgatttatt aattaccagg
atccttttct aaattgaatg tacttttgaa ttactaggtt 1980tcttcttttt
ttttgttctg caatagtgaa agaaaactca gtagtttagt ttcagtttct
2040catggaaatt ggtaaatgtt agttttgact tcatctattt tttatttgtt
tttattagcg 2100tagagtagga agtctcatat tctactgttc tatctaggat
ggtgaaattc caaaggtgcc 2160taacttgagt aagggatttg tgacaagata
gtacacatta ctataagggc tattatttcc 2220tgaactggat gtccctaaaa
gcaaataaac tgcccactat ctct 226421205PRTHomo sapiens 21Met Glu Gly
Leu Ser Asp Val Ala Ser Phe Ala Thr Lys Leu Lys Asn1 5 10 15Thr Leu
Ile Gln Tyr His Ser Ile Glu Glu Asp Lys Trp Arg Val Ala 20 25 30Lys
Lys Thr Lys Asp Val Thr Val Trp Arg Lys Pro Ser Glu Glu Phe 35 40
45Asn Gly Tyr Leu Tyr Lys Ala Gln Gly Val Ile Asp Asp Leu Val Tyr
50 55 60Ser Ile Ile Asp His Ile Arg Pro Gly Pro Cys Arg Leu Asp Trp
Asp65 70 75 80Ser Leu Met Thr Ser Leu Asp Ile Leu Glu Asn Phe Glu
Glu Asn Cys 85 90 95Cys Val Met Arg Tyr Thr Thr Ala Gly Gln Leu Trp
Asn Ile Ile Ser 100 105 110Pro Arg Glu Phe Val Asp Phe Ser Tyr Thr
Val Gly Tyr Lys Glu Gly 115 120 125Leu Leu Ser Cys Gly Ile Ser Leu
Asp Trp Asp Glu Lys Arg Pro Glu 130 135 140Phe Val Arg Gly Tyr Asn
His Pro Cys Gly Trp Phe Cys Val Pro Leu145 150 155 160Lys Asp Asn
Pro Asn Gln Ser Leu Leu Thr Gly Tyr Ile Gln Thr Asp 165 170 175Leu
Arg Gly Met Ile Pro Gln Ser Ala Val Asp Thr Ala Met Ala Ser 180 185
190Thr Leu Thr Asn Phe Tyr Gly Asp Leu Arg Lys Ala Leu 195 200
205221344DNAHomo sapiens 22gagctccagc ctccaggcac ccgggatcca
gcgccgccgc tcataacacc cgcgaccccg 60cagctaagcg cagctcccga cgcaatggac
ccggcgctgg cagcccagat gagcgaggct 120gtggccgaga agatgctcca
gtaccggcgg gacacagcag gctggaagat ttgccgggaa 180ggcaatggag
tttcagtttc ctggaggcca tctgtggagt ttccagggaa cctgtaccga
240ggagaaggca ttgtatatgg gacactagag gaggtgtggg actgtgtgaa
gccagctgtt 300ggaggcctac gagtgaagtg ggatgagaat gtgaccggtt
ttgaaattat ccaaagcatc 360actgacaccc tgtgtgtaag cagaacctcc
actccctccg ctgccatgaa gctcatttct 420cccagagatt ttgtggactt
ggtgctagtc aagagatatg aggatgggac catcagttcc 480aacgccaccc
atgtggagca tccgttatgt cccccgaagc caggttttgt gagaggattt
540aaccatcctt gtggttgctt ctgtgaacct cttccagggg aacccaccaa
gaccaacctg 600gtcacattct tccataccga cctcagcggt tacctcccac
agaacgtggt ggactccttc 660ttcccccgca gcatgacccg gttttatgcc
aaccttcaga aagcagtgaa gcaattccat 720gagtaatgct atcgttactt
cttggcaaag aactcccgtg actcatcgag gagctccagc 780tgttgggaca
ccaaggagcc tgggagcacg cagaggcctg tgttcactct ttggaacaag
840ctgatggact gcgcatctct gagaatgcca accagaggcg gcagcccacc
cttcctgcct 900cctgccccac tcagggttgg cgtgtgatga gccattcatg
tgttccaaac tccatctgcc 960tgttacccaa acacgcctct cctggcaggg
tagacccagg cctctaacca tctgacagag 1020actcggcctg gacaccatgc
gatgcactct ggcaccaagg ctttatgtgc ccatcactct 1080cagagaccac
gtttccctga ctgtcataga gaatcatcat cgccactgaa aaccaggccc
1140tgttgccttt taagcatgta ccgctccctc agtcctgtgc tgcagccccc
caaatatatt 1200tttctgatat agaccttgta tatggcttta atgccgcaaa
atatttattt ttccttaaaa 1260aaggtgtcaa cttggaaata atggtttaaa
aacaggataa gcattaagga aaaacaaaaa 1320aaaaaaaaaa aaaaaaaaaa aaaa
134423213PRTHomo sapiens 23Met Asp Pro Ala Leu Ala Ala Gln Met Ser
Glu Ala Val Ala Glu Lys1 5 10 15Met Leu Gln Tyr Arg Arg Asp Thr Ala
Gly Trp Lys Ile Cys Arg Glu 20 25 30Gly Asn Gly Val Ser Val Ser Trp
Arg Pro Ser Val Glu Phe Pro Gly 35 40 45Asn Leu Tyr Arg Gly Glu Gly
Ile Val Tyr Gly Thr Leu Glu Glu Val 50 55 60Trp Asp Cys Val Lys Pro
Ala Val Gly Gly Leu Arg Val Lys Trp Asp65 70 75 80Glu Asn Val Thr
Gly Phe Glu Ile Ile Gln Ser Ile Thr Asp Thr Leu 85 90 95Cys Val Ser
Arg Thr Ser Thr Pro Ser Ala Ala Met Lys Leu Ile Ser 100 105 110Pro
Arg Asp Phe Val Asp Leu Val Leu Val Lys Arg Tyr Glu Asp Gly 115 120
125Thr Ile Ser Ser Asn Ala Thr His Val Glu His Pro Leu Cys Pro Pro
130 135 140Lys Pro Gly Phe Val Arg Gly Phe Asn His Pro Cys Gly Cys
Phe Cys145 150 155 160Glu Pro Leu Pro Gly Glu Pro Thr Lys Thr Asn
Leu Val Thr Phe Phe 165 170 175His Thr Asp Leu Ser Gly Tyr Leu Pro
Gln Asn Val Val Asp Ser Phe 180 185 190Phe Pro Arg Ser Met Thr Arg
Phe Tyr Ala Asn Leu Gln Lys Ala Val 195 200 205Lys Gln Phe His Glu
21024663DNAHomo sapiens 24atggacttca aggcaattgc ccaacaaact
gcccaagaag ttttaggtta taatcgagat 60acatcaggct ggaaagtggt taaaacttca
aaaaagataa ctgtttccag taaggcttct 120agaaaattcc atggaaatct
atatcgtgtt gaagggataa ttccagaatc accagctaaa 180ctatctgatt
tcctctacca aactggagac agaattacat gggataaatc attgcaagtg
240tataatatgg tacacaggat tgattcggac acattcatat gtcataccat
tacacaaagt 300tttgccgtgg gctccatttc ccctcgagac tttatcgact
tagtgtacat caagcgctac 360gaaggaaata tgaacattat cagttctaaa
agtgtggatt ttccagaata tcctccatct 420tcaaattata tccgcggtta
taaccatcct tgtggctttg tatgttcacc aatggaagaa 480aacccagcat
attccaaact agtgatgttt gtccagacag aaatgagagg aaaattgtcc
540ccatcaataa ttgaaaaaac catgccttcc aacttagtaa acttcatcct
caatgcaaaa 600gatggaataa aggcacacag aactccatca agacgtggat
ttcatcataa tagtcattca 660tga 66325220PRTHomo sapiens 25Met Asp Phe
Lys Ala Ile Ala Gln Gln Thr Ala Gln Glu Val Leu Gly1 5 10 15Tyr Asn
Arg Asp Thr Ser Gly Trp Lys Val Val Lys Thr Ser Lys Lys 20 25 30Ile
Thr Val Ser Ser Lys Ala Ser Arg Lys Phe His Gly Asn Leu Tyr 35 40
45Arg Val Glu Gly Ile Ile Pro Glu Ser Pro Ala Lys Leu Ser Asp Phe
50 55 60Leu Tyr Gln Thr Gly Asp Arg Ile Thr Trp Asp Lys Ser Leu Gln
Val65 70 75 80Tyr Asn Met Val His Arg Ile Asp Ser Asp Thr Phe Ile
Cys His Thr 85 90 95Ile Thr Gln Ser Phe Ala
Val Gly Ser Ile Ser Pro Arg Asp Phe Ile 100 105 110Asp Leu Val Tyr
Ile Lys Arg Tyr Glu Gly Asn Met Asn Ile Ile Ser 115 120 125Ser Lys
Ser Val Asp Phe Pro Glu Tyr Pro Pro Ser Ser Asn Tyr Ile 130 135
140Arg Gly Tyr Asn His Pro Cys Gly Phe Val Cys Ser Pro Met Glu
Glu145 150 155 160Asn Pro Ala Tyr Ser Lys Leu Val Met Phe Val Gln
Thr Glu Met Arg 165 170 175Gly Lys Leu Ser Pro Ser Ile Ile Glu Lys
Thr Met Pro Ser Asn Leu 180 185 190Val Asn Phe Ile Leu Asn Ala Lys
Asp Gly Ile Lys Ala His Arg Thr 195 200 205Pro Ser Arg Arg Gly Phe
His His Asn Ser His Ser 210 215 220261932DNAHomo sapiens
26ccggactgcg gaaggatgga gctggccgcc ggaagcttct cggaggagca gttctgggag
60gcctgcgccg agctccagca gcccgctctg gccggggccg actggcagct cctagtggag
120acctcgggca tcagcatcta ccggctgctg gacaagaaga ctggacttca
tgagtataaa 180gtctttggtg ttctggagga ctgctcacca actctactgg
cagacatcta tatggactca 240gattacagaa aacaatggga ccagtatgtt
aaagaactct atgaacaaga atgcaacgga 300gagactgtgg tctactggga
agtgaagtac ccttttccca tgtccaacag agactatgtc 360taccttcggc
agcggcgaga cctggacatg gaagggagga agatccatgt gatcctggcc
420cggagcacct ccatgcctca gcttggcgag aggtctgggg tgatccgggt
gaagcaatac 480aagcagagcc tggcgattga gagtgacggc aagaagggga
gcaaagtttt catgtattac 540ttcgataacc cgggtggcca aattccgtcc
tggctcatta actgggccgc caagaatgga 600gttcctaact tcttgaaaga
catggcaaga gcctgtcaga actacctcaa gaaaacctaa 660gaaagagaac
tgggaacatt gcatccatgg gttgatgtct ctggaagtgc aaccacccaa
720tgtctctgga agtgccacct ggaagtgcca cctggaagtg tctctggaag
agcacccacc 780actgttcagc cttcccctgc tgtttctgtc ttcagaggcc
tacacactac cacatccttt 840ctaagcatgt ttgcctgaca tccagctcac
tcgtctgctt cctttctcgc tccccccatc 900ctgggctggg ctgccttctt
ctacagttca atatggggca gactagggaa acctttgctt 960gcttactatt
aggaggggaa gtcttcagta gggaacacga tcattccatt gtgcaatttt
1020acggggatgg gtgggcggag ggacacaaca aaatttaaga atgactattt
gggcgggctg 1080gctcttttgc agcttgtgat ttcttccagc ttgggagggg
ctgctggaag tggcatttcg 1140ttcagagctg actttcagtg cacccaaact
ggatgacgtg ccaatgtcca tttgccttat 1200gctttgtgga gctgattagg
ctgggatttg aggtgataat ccagtaagtc tttcctcgtt 1260cctacttgtg
gaggatcagt agctgttatg atgccagacc atttggagaa gtatcagagg
1320cctgaccgga cacataatac gacaaccaca tttttcctca tcatccatga
ggaaatggat 1380gatttctctt ttccatatgt cactggggga aaggctgcct
gtacctctca agctttgcat 1440tttactggaa actgaggcgt caagatggct
gtggcagcta gcaaaagcaa agatgctttg 1500tgcatagcct tgtgaaaaag
tatctttcta tgcaataaga tgaattttcc tcccagaata 1560tttagaaatg
tagaagggat aacagttcac agccaggtaa aatttaactg gtggcttaat
1620gactctgcac ctttttctca ggaattctgc ctaagttgtc tgccttttct
accaccaaaa 1680agacttttag ttttctatgc tttctcctga attttggtag
ggtaagtatt tctatgtcaa 1740aggcacagcc ttgatgatct cagggaaaaa
ttttaatcac tgtgtataat gatactgaac 1800cttgattaat aacagaaatt
caggatgtaa agccacagaa tgggatttat taatgtggga 1860tacctcagac
tgtttgtttt ctttctggga agaaaagtgt gttctataat gaataaatat
1920agagtggttt tt 193227214PRTHomo sapiens 27Met Glu Leu Ala Ala
Gly Ser Phe Ser Glu Glu Gln Phe Trp Glu Ala1 5 10 15Cys Ala Glu Leu
Gln Gln Pro Ala Leu Ala Gly Ala Asp Trp Gln Leu 20 25 30Leu Val Glu
Thr Ser Gly Ile Ser Ile Tyr Arg Leu Leu Asp Lys Lys 35 40 45Thr Gly
Leu His Glu Tyr Lys Val Phe Gly Val Leu Glu Asp Cys Ser 50 55 60Pro
Thr Leu Leu Ala Asp Ile Tyr Met Asp Ser Asp Tyr Arg Lys Gln65 70 75
80Trp Asp Gln Tyr Val Lys Glu Leu Tyr Glu Gln Glu Cys Asn Gly Glu
85 90 95Thr Val Val Tyr Trp Glu Val Lys Tyr Pro Phe Pro Met Ser Asn
Arg 100 105 110Asp Tyr Val Tyr Leu Arg Gln Arg Arg Asp Leu Asp Met
Glu Gly Arg 115 120 125Lys Ile His Val Ile Leu Ala Arg Ser Thr Ser
Met Pro Gln Leu Gly 130 135 140Glu Arg Ser Gly Val Ile Arg Val Lys
Gln Tyr Lys Gln Ser Leu Ala145 150 155 160Ile Glu Ser Asp Gly Lys
Lys Gly Ser Lys Val Phe Met Tyr Tyr Phe 165 170 175Asp Asn Pro Gly
Gly Gln Ile Pro Ser Trp Leu Ile Asn Trp Ala Ala 180 185 190Lys Asn
Gly Val Pro Asn Phe Leu Lys Asp Met Ala Arg Ala Cys Gln 195 200
205Asn Tyr Leu Lys Lys Thr 21028271PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
sequence 28Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa85 90 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa100 105 110Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa115 120 125Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg130 135 140Xaa
Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa145 150
155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Arg180 185 190Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa195 200 205Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa210 215 220Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa225 230 235 240Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa245 250 255Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa260 265
27029271PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 29Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Arg 130 135 140Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ser Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg 180 185 190Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa225 230
235 240Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 245 250 255Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 260 265 27030271PRTArtificial SequenceDescription of
Artificial Sequence Synthetic consensus sequence 30Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa
Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa 50 55
60Xaa Xaa Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65
70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Asp Xaa Xaa Xaa Xaa
Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 100 105 110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Pro Xaa Xaa Pro Xaa Arg 130 135 140Asp Xaa Val Xaa Val Arg Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa 165 170 175Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg 180 185 190Xaa
Xaa Xaa Xaa Pro Ser Gly Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Gly 195 200
205Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
210 215 220Xaa Xaa Xaa Xaa Xaa Asp Leu Xaa Gly Xaa Xaa Pro Xaa Xaa
Xaa Xaa225 230 235 240Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 245 250 255Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Leu
Xaa Xaa Xaa Xaa Xaa Xaa 260 265 27031202PRTRattus sp. 31His Phe Leu
Gln Asp Cys Val Asp Gly Leu Phe Lys Glu Val Lys Glu1 5 10 15Lys Phe
Lys Gly Trp Val Ser Tyr Pro Thr Ser Glu Gln Ala Glu Leu 20 25 30Ser
Tyr Lys Lys Val Ser Glu Gly Pro Pro Leu Arg Leu Trp Arg Ala 35 40
45Thr Ile Glu Val Pro Ala Ala Pro Glu Glu Ile Leu Lys Arg Leu Leu
50 55 60Lys Glu Gln His Leu Trp Asp Val Asp Leu Leu Asp Ser Lys Val
Ile65 70 75 80Glu Ile Leu Asp Ser Gln Thr Glu Ile Tyr Gln Tyr Val
Gln Asn Ser 85 90 95Met Ala Pro His Pro Ala Arg Asp Tyr Val Val Leu
Arg Thr Trp Arg 100 105 110Thr Asn Leu Pro Arg Gly Ala Cys Ala Leu
Leu Phe Thr Ser Val Asp 115 120 125His Asp Arg Ala Pro Val Ala Gly
Val Arg Val Asn Val Leu Leu Ser 130 135 140Arg Tyr Leu Ile Glu Pro
Cys Gly Ser Gly Lys Ser Lys Leu Thr Tyr145 150 155 160Met Cys Arg
Ala Asp Leu Arg Gly His Met Pro Glu Trp Tyr Thr Lys 165 170 175Ser
Phe Gly His Leu Cys Ala Ala Glu Val Val Lys Ile Arg Asp Ser 180 185
190Phe Ser Asn Gln Ser Thr Glu Ser Lys Asp 195 200326PRTHomo
sapiens 32Leu Arg Arg His Gly Ser1 5336PRTHomo sapiens 33Val Lys
Arg Glu Asp Ser1 5
* * * * *