U.S. patent application number 11/791989 was filed with the patent office on 2008-08-28 for rhamnose promoter expression system.
Invention is credited to Johann Brass, Christoph Kiziak, Joachim Klein, Ralf Ostendorp.
Application Number | 20080206817 11/791989 |
Document ID | / |
Family ID | 36304177 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080206817 |
Kind Code |
A1 |
Brass; Johann ; et
al. |
August 28, 2008 |
Rhamnose Promoter Expression System
Abstract
Vectors expressible in a host that is the rhaBAD promoter region
of the L-rhamnose operon operably linked to a transcriptional unit
that is: a) a nucleic acid sequence which is heterologouse to the
host, and b) a prokaryotic signal sequence operably linked to the
nucleic acid sequence. The prokaryotic signal sequence is selected
from signal peptides of periplasmatic binding proteins for sugars,
amino acids, vitamins and ions. The expression of the nucleic acid
sequence is controlled by the promoter region. The vector is used
for the regulated heterologous expression of a nucleic acid
sequence in a prokaryotic host. This is an isolated and purified
nucleic acid sequence expressible in a host is the promoter region
of the L-rhamnose operon. There is a method for producing a
polypeptide in a host using the vector.
Inventors: |
Brass; Johann; (Ausserberg,
CH) ; Kiziak; Christoph; (Visp, CH) ; Klein;
Joachim; (Munchen, CH) ; Ostendorp; Ralf;
(Munchen, DE) |
Correspondence
Address: |
FISHER, CHRISTEN & SABOL
1725 K STREET, N.W., SUITE 1108
WASHINGTON
DC
20006
US
|
Family ID: |
36304177 |
Appl. No.: |
11/791989 |
Filed: |
December 5, 2005 |
PCT Filed: |
December 5, 2005 |
PCT NO: |
PCT/EP2005/013013 |
371 Date: |
August 28, 2007 |
Current U.S.
Class: |
435/69.6 ;
435/243; 435/320.1; 435/471; 435/69.1; 536/23.1 |
Current CPC
Class: |
C07K 2317/14 20130101;
C12N 15/70 20130101; C07K 16/00 20130101; C07K 2317/622 20130101;
C07K 2317/55 20130101; C12N 15/78 20130101; C12N 15/63
20130101 |
Class at
Publication: |
435/69.6 ;
435/320.1; 435/471; 536/23.1; 435/243; 435/69.1 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C12N 15/63 20060101 C12N015/63; C12N 15/00 20060101
C12N015/00; C07H 21/00 20060101 C07H021/00; C12N 1/00 20060101
C12N001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 7, 2004 |
EP |
04028917.5 |
Claims
1. A vector expressible in a host comprising the rhaBAD promoter
region of the L-rhamnose operon operably linked to a
transcriptional unit comprising: a) a nucleic acid sequence which
is heterologous to said host, and b) a prokaryotic signal sequence
operably linked to said nucleic acid sequence, whereas said
prokaryotic signal sequence is selected from a group consisting of
signal peptides of periplasmatic binding proteins for sugars, amino
acids, vitamins and ions and, whereas the expression of said
nucleic acid sequence is controlled by said promoter region.
2. The vector of claim 1, wherein said promoter region is the
rhaBAD promoter.
3. The vector of claim 2, wherein said rhaBAD promoter consists of
the sequence SEQ ID NO. 1, a sequence complementary thereof and
variants thereof.
4. The vector of claim 3, wherein said signal peptides are selected
from the group consisting of periplasmatic binding proteins for
sugars, amino acids, vitamins and ions, are E. coli signal peptides
selected from the group consisting of LamB (Maltoporin precursor),
MalE (Maltose-binding protein precursor), Bla (Beta-lactamase),
OppA (periplasmic oligopeptide-binding protein), TreA (periplasmic
trehalase precursor), MppA (periplasmic murein peptide-binding
protein precursor), BglX (Periplasmic beta-glucosidase precursor),
ArgT (Lysinearginine-ornithine binding periplasmic protein
precursor), MalS (Alpha-amylase precursor), HisJ (Histidine-binding
periplasmic protein precursor), XylF (D-Xylose-binding periplasmic
protein precursor), FecB (dicitrate-binding periplasmic protein
precursor), OmpA (outer membrane protein A precursor) and PhoA
(Alkaline phosphatase precursor).
5. The vector of claim 4, wherein said transcriptional unit further
comprises, a translation initiation region upstream of the
initiation point of the translation of said transcriptional unit,
said translation initiation region consisting of the sequence
AGGAGATATACAT (SEQ ID NO. 2), whereas said translation initiation
region is operably linked to said nucleic acid sequence.
6. The vector of claim 5, wherein said transcriptional unit further
comprises a transcription termination region which is rrnB
transcriptional terminator sequence.
7. The vector of claim 6, wherein said nucleic acid sequence
encodes a polypeptide.
8. The vector of claim 6, wherein said nucleic acid sequence
encodes an antibody.
9. The vector of claim 6, wherein said nucleic acid sequence
encodes a Fab fragment.
10. The vector of claim 9, wherein the heavy and light chain of
said Fab fragment are encoded by a dicistronic transcriptional
unit, whereas each chain is operably linked to said prokaryotic
signal sequence and an identical translation initiation region
upstream of the initiation point of the translation of said
transcriptional unit.
11. The vector of claim 10, wherein said rhaBAD promoter region and
said operably linked transcriptional unit consists of sequence SEQ
ID NO. 3, a sequence complementary thereof and variants
thereof.
12. The vector of claim 10, wherein said rhaBAD promoter region and
said operably linked transcriptional unit consists of sequence SEQ
ID NO. 4, a sequence complementary thereof and variants
thereof.
13. The vector of claim 12, wherein said vector is an autonomously
or self-replicating plasmid, a cosmid, a phage, a virus or a
retrovirus.
14. A process for utilizing the vector of claim 13, for regulated
heterologous expression of a nucleic acid sequence in a prokaryotic
host.
15. The process of utilizing the vector of claim 14, wherein said
nucleic acid sequence encodes for a polypeptide.
16. The process of utilizing the vector of claim 15, wherein said
polypeptide is a Fab fragment, whereas heavy and light chains of
the Fab fragment are expressed in equal amounts.
17. An isolated and purified nucleic acid sequence expressible in a
host comprising rhaBAD promoter region of L-rhamnose operon
operably linked to a transcriptional unit comprising: a) a nucleic
acid sequence which is heterologous to said host, and b) a
prokaryotic signal sequence operably linked to said nucleic acid
sequence, whereas said prokaryotic signal sequence is selected from
the group consisting of signal peptides of periplasmatic binding
proteins for sugars, amino acids, vitamins and ions and, whereas
the expression of said nucleic acid sequence is controlled by said
promoter region.
18. The isolated and purified nucleic acid sequence of claim 17,
wherein said promoter region is the rhaBAD promoter.
19. The isolated and purified nucleic acid sequence of claim 18,
wherein said rhaBAD promoter consists of sequence SEQ ID NO. 1, a
sequence complementary thereof and variants thereof.
20. The isolated and purified nucleic acid sequence of claim 19,
wherein said rhaBAD promoter region and said operably linked
transcriptional unit consists of sequence SEQ ID NO. 3, a sequence
complementary thereof and variants thereof.
21. The isolated and purified nucleic acid sequence of claim 19,
wherein said rhaBAD promoter region and said operably linked
transcriptional unit consists of sequence SEQ ID NO. 4, a sequence
complementary thereof and variants thereof.
22. Plasmid pBW22-Fab-H.
23. Plasmid pAKL14.
24. A prokaryotic host transformed with the vector of claim 13.
25. A prokaryotic host transformed with the isolated and purified
nucleic acid sequence of claim 21.
26. A prokaryotic host transformed with the plasmids of claim
23.
27. A method for producing a polypeptide in a host, comprising the
steps of: a) constructing a vector of claim 13, b) transforming a
prokaryotic host with said vector, c) allowing expression of said
polypeptide in a cell culture system under suitable conditions, and
d) recovering said polypeptide from the cell culture system.
28. The method of claim 27, whereas the polypeptide produced is a
Fab fragment, whereas heavy and light chains of the Fab fragment
are expressed in said cell culture system in equal amounts.
29. The method of claim 28, whereas expression of said polypeptide
is carried out in glycerol containing medium.
30. A vector expressible in a host comprising a promoter region
operably linked to a transcriptional unit comprising: a) a nucleic
acid sequence which is heterologous to said host, and b) a
translation initiation region upstream of initiation point of the
translation of said transcriptional unit, said translation
initiation region consisting of sequence AGGAGATATACAT (SEQ ID NO.
2), whereas said translation initiation region is operably linked
to said nucleic acid sequence and the expression of said nucleic
acid sequence is controlled by said promoter region.
31. The vector of claim 30, wherein said promoter region is rhaBAD
promoter region of L-rhamnose operon.
32. The vector of claim 31, wherein said transcriptional unit
further comprises a signal sequence operably linked to said nucleic
acid sequence.
33. The vector of claim 32, wherein said nucleic acid sequence
encodes a polypeptide.
34. The vector of claim 32, wherein said nucleic acid sequence
encodes an antibody.
35. The vector of claim 32, wherein said nucleic acid sequence
encodes a Fab fragment.
36. The vector of claim 35, wherein heavy and the light chain of
said Fab fragment are encoded by a dicistronic transcriptional
unit, whereas each chain is operably linked to said signal sequence
and said translation initiation region.
37. A method for producing a polypeptide in a host, comprising the
steps of: a) constructing a vector of claim 36, b) transforming a
prokaryotic host with said vector, c) allowing expression of said
polypeptide in a cell culture system under suitable conditions, and
d) recovering said polypeptide from the cell culture system.
38. The method of claim 37, whereas the polypeptide produced is a
Fab fragment, whereas heavy and light chains of the Fab fragment
are expressed in said cell culture system in equal amounts.
39. The vector of claim 1, wherein said signal peptides is selected
from the group consisting of periplasmatic binding proteins for
sugars, amino acids, vitamins and ions, are E. coli signal peptides
selected from the group consisting of LamB (Maltoporin precursor),
MalE (Maltose-binding protein precursor), Bla (Beta-lactamase),
OppA (periplasmic oligopeptide-binding protein), TreA (periplasmic
trehalase precursor), MppA (periplasmic murein peptide-binding
protein precursor), BglX (Periplasmic beta-glucosidase precursor),
ArgT (Lysinearginine-ornithine binding periplasmic protein
precursor), MalS (Alpha-amylase precursor), HisJ (Histidine-binding
periplasmic protein precursor), XylF (D-Xylose-binding periplasmic
protein precursor), FecB (dicitrate-binding periplasmic protein
precursor), OmpA (outer membrane protein A precursor) and PhoA
(Alkaline phosphatase precursor).
40. The vector of claim 1, wherein said transcriptional unit
further comprises, a translation initiation region upstream of
initiation point of the translation of said transcriptional unit,
said translation initiation region consisting of sequence
AGGAGATATACAT (SEQ ID NO. 2), wherein said translation initiation
region is operably linked to said nucleic acid sequence.
41. The vector of claim 1, wherein said transcriptional unit
further comprises a transcription termination region which is the
rrnB transcriptional terminator sequence.
42. The vector of claim 1, wherein said nucleic acid sequence
encodes a polypeptide.
43. The vector of claim 1, wherein said nucleic acid sequence
encodes an antibody.
44. The vector of claim 1, wherein said nucleic acid sequence
encodes a Fab fragment.
45. The vector of claim 44, wherein heavy and light chain of said
Fab fragment are encoded by a dicistronic transcriptional unit,
whereas each chain is operably linked to said prokaryotic signal
sequence and an identical translation initiation region upstream of
initiation point of the translation of said transcriptional
unit.
46. The vector of claim 1, wherein said rhaBAD promoter region and
said operably linked transcriptional unit consists of sequence SEQ
ID NO. 3, a sequence complementary thereof and variants
thereof.
47. The vector of claim 1, wherein said rhaBAD promoter region and
said operably linked transcriptional unit consists of sequence SEQ
ID NO. 4, a sequence complementary thereof and variants
thereof.
48. The vector of claim 1, wherein said vector is an autonomously
or self-replicating plasmid, a cosmid, a phage, a virus or a
retrovirus.
49. A process for utilizing the vector of claim 1, for the
regulated heterologous expression of a nucleic acid sequence in a
prokaryotic host.
50. The isolated and purified nucleic acid sequence of claim 17,
wherein said rhaBAD promoter region and said operably linked
transcriptional unit consists of sequence SEQ ID NO. 3, a sequence
complementary thereof and variants thereof.
51. The isolated and purified nucleic acid sequence of claim 17,
wherein said rhaBAD promoter region and said operably linked
transcriptional unit consists of sequence SEQ ID NO. 4, a sequence
complementary thereof and variants thereof.
52. A prokaryotic host transformed with the vector of claim 1.
53. A prokaryotic host transformed with the isolated and purified
nucleic acid sequence of claim 17.
54. A prokaryotic host transformed with the plasmids of claim
22.
55. A method for producing a polypeptide in a host, comprising the
steps of: a) constructing a vector of claim 7, b) transforming a
prokaryotic host with said vector, c) allowing expression of said
polypeptide in a cell culture system under suitable conditions, and
d) recovering said polypeptide from the cell culture system.
56. The method of claim 55, wherein expression of said polypeptide
is carried out in glycerol containing medium.
57. The vector of claim 30, wherein said transcriptional unit
further comprises a signal sequence operably linked to said nucleic
acid sequence.
58. The vector of claim 30, wherein said nucleic acid sequence
encodes a polypeptide.
59. The vector of claim 30, wherein said nucleic acid sequence
encodes an antibody.
60. The vector of claim 30, wherein said nucleic acid sequence
encodes a Fab fragment.
61. A method for producing a polypeptide in a host, comprising the
steps of: a) constructing a vector of claim 30, b) transforming a
prokaryotic host with said vector, c) allowing expression of said
polypeptide in a cell culture system under suitable conditions, and
d) recovering said polypeptide from the cell culture system
Description
[0001] The present invention concerns vectors for the heterologous
expression of nucleic acids encoding e. g. polypeptides such as
recombinant proteins in prokaryotic hosts. More specifically, the
present invention relates to new vectors expressible in a host
comprising the rhaBAD promoter region of the L-rhamnose operon
operably linked to a transcriptional unit comprising [0002] a) a
nucleic acid sequence which is heterologous to said host [0003] b)
a prokaryotic signal sequence operably linked to said nucleic acid
sequence, whereas said prokaryotic signal sequence is selected from
signal peptides of periplasmatic binding proteins for sugars, amino
acids, vitamins and ions and, whereas the expression of said
nucleic acid sequence is controlled by said promoter region.
Furthermore the invention relates to the use of these vectors for
the heterologous expression of nucleic acids encoding e. g.
polypeptides.
BACKGROUND OF THE INVENTION
[0004] Many systems have been described for the heterologous
expression of nucleic acids encoding e. g. polypeptides such as
recombinant proteins in prokaryotic systems. However, most
heterologous gene expression systems in prokaryotic host systems
have relied exclusively on a limited set of bacterial promoters.
The most widely used prokaryotic promoters have included the
lactose [lac] (Yanisch-Perron et al., 1985, Gene 33, 103-109), and
the tryptophan [trp] (Goeddel et al., 1980, Nature (London) 287,
411-416) promoters, and the hybrid promoters derived from these two
[tac and trc] (Brosius, 1984, Gene 27:161-172; Amann and Brosius,
1985, Gene 40,183-190). Other inducible promoter systems such as
the araB promoter inducible by arabinose (WO 86 04356), the
rhamnose promoter rhaSB inducible by L-rhamnose (WO 03068956) or
the rhamnose promoter rhaBAD inducible by L-rhamnose (WO
2004/050877) have been described as well for the heterologous
expression of proteins. WO 2004/050877 describes the use of a
rhaBAD promoter for the heterologous expression of nitrilase in E.
coli. After induction with L-rhamnose, nitrilase activity in
resting-cell assays could be obtained. However, in particular for
the expression of complex polypeptides such as antibodies or
antibody fragments it is advantageous to export the polypeptide
from the cytoplasma to non-cytoplasmic locations (secretion) by
using signal sequences, since the overproduction of heterologous
proteins in the cytoplasm is often accompanied by the misfolding
and segregation into insoluble aggregates (inclusion bodies).
However, since the signal sequence can influence secondary and
tertiary structure formation in the mature region of secretory
polypeptides, the choice of the appropriate signal sequence in
combination with an useful promoter is important for high
production of functional polypeptides. Thus, there is a need to
provide alternative prokaryotic expression systems for the
heterologous expression of nucleic acid sequences.
SUMMARY OF THE INVENTION
[0005] These and other objects as will be apparent from the
foregoing description have been achieved by providing new vectors,
which are useful for high-level expression of a desired
heterologous product, and which comprise the rhaBAD promoter region
of the L-rhamnose operon, a heterologous nucleic acid sequence and
a prokaryotic signal sequence selected from signal peptides of
periplasmatic binding proteins for sugars, amino acids, vitamins
and ions. In a first aspect, the object of the present invention is
to provide a new vector expressible in a host comprising the rhaBAD
promoter region of the L-rhamnose operon operably linked to a
transcriptional unit comprising [0006] a) a nucleic acid sequence
which is heterologous to said host [0007] b) a prokaryotic signal
sequence operably linked to said nucleic acid sequence, whereas
said prokaryotic signal sequence is selected from signal peptides
of periplasmatic binding proteins for sugars, amino acids, vitamins
and ions and, whereas the expression of said nucleic acid sequence
is controlled by said promoter region. Also provided are: the use
of said new vector for the regulated heterologous expression of a
nucleic acid sequence in a prokaryotic host; an isolated and
purified nucleic acid sequence expressible in a host comprising the
rhaBAD promoter region of the L-rhamnose operon, a heterologous
nucleotide sequence and a prokaryotic signal sequence selected from
signal peptides of periplasmatic binding proteins for sugars, amino
acids, vitamins and ions; a prokaryotic host transformed with said
vector or said isolated and purified nucleic acid sequence; a
method for producing a polypeptide in a host using said vector; and
a vector comprising a promoter region, a heterologous nucleic acid
sequence and a translation initiation region consisting of the
sequence AGGAGATATACAT.
[0008] Other objects and advantages will become apparent to those
skilled in the art from a review of the ensuing detailed
description, which proceeds with reference to the following
illustrative drawings, and the attendant claims.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 shows plasmid pBW22-Fab-H containing the L-rhamnose
inducible promoter (PrhaBAD), sequences coding for signal sequences
operably linked to the light chain (ompA-VL3-CL) and the heavy
chain (phoA-VH-CH) of a Fab fragment, and a transcription
termination region (rrnB).
[0010] FIG. 2 shows plasmid pBLL15 containing a melibiose inducible
promoter (PmelAB2), sequences coding for signal sequences operably
linked to the light chain (ompA-VL3-CL) and the heavy chain
(phoA-VH-CH) of a Fab fragment, and a transcription termination
region (rrnB).
[0011] FIG. 3 shows dotblot results (with anti-human light chain
for detecting Fab, alkaline peroxidase conjugated) of lysozyme
extracts of the uminduced (-) and induced (+) W3110 strains with
the different expression plasmids. The time intervals are
indicated.
[0012] FIG. 4 shows plasmid pAKL14 containing the L-rhamnose
inducible promoter (PrhaBAD) and the Fab-H genes with altered
signal sequences.
[0013] FIG. 5 shows dot blot of lysozyme extracts of uninduced (-)
and L-rhamnose induced strain W3110 (pAKL14). The time when samples
were taken is indicated (with anti-human light chain for detecting
Fab, alkaline peroxidase conjugated).
[0014] FIG. 6 shows a Western blot of lysozyme extracts of
L-rhamnose induced strain W3110 (pAKL14). The time after induction
when the samples were taken is indicated (with anti-human light
chain for detecting Fab, alkaline peroxidase conjugated). Lane 1:
Standard (1.28 .mu.g); lane 2: W3110 (pAKL14), ind., 3 h; lane 3:
W3110 (pAKL14), ind., 5 h; lane 4: W3110 (pAKL14), ind., 7 h; lane
5: W3110 (pAKL14), ind., 12 h; lane 6: W3110 (pAKL14), ind., 23 h;
lane 7: W3110 (pAKL14), not ind., 23 h.
[0015] FIG. 7 shows SDS-PAGE of lysozyme extracts of different
W3110 strains with high Fab-H antibody concentrations. The strains
producing the light and heavy chain without signal sequences are
used as a negative reference (lane 1: Marker; lane 2: W3110
(pMx9-HuCAL-Fab-H); lane 3: W3110 (pBW22-Fab-H); lane 4: W3110
(pBLL15), lane 5: W3110 (pAKL14); lane 6: Standard (2 .mu.g)).
[0016] FIG. 8 shows plasmid pAKL15E containing the melibiose
inducible promoter (PmelAB2) and the Fab-H genes with altered
signal sequences.
[0017] FIG. 9: shows SDS-PAGE of lysozyme extracts of strain W3110
(pAKL15E) in the presence or absence of the inducer melibiose. The
position of the light and heavy chain is indicated (lane 1: Marker;
lane 2: W3110 (pAKL15E), not induced; lane 3: W3110 (pAKL15E),
induced).
[0018] FIG. 10 shows plasmid pBW22-pelB-S1 comprising the
L-rhamnose inducible rhaBAD promoter, a sequence coding for a PelB
signal peptide operably linked to a sequence coding for a single
chain antibody (scFv, S1), and a transcription termination region
(rrnB).
[0019] FIG. 11 shows SDS-PAGE for crude extracts of not induced (-)
and induced (+) strain W3110 (pBW22-pelB-S1). Samples were taken
after different time intervals as indicated. The soluble and
insoluble protein fractions after lysozyme treatment were analyzed.
An arrow indicates the scFv protein. M=Mark12, molecular weight
standard of Invitrogen.
[0020] FIG. 12 shows the broad-host-range plasmid pJOE4782
comprising the L-rhamnose inducible rhaBAD promoter in combination
with the genes of the regulatory proteins RhaS and RhaR of the
L-rhamnose operon of Escherichia coli. Plasmid pJOE4782 further
contains a sequence coding for a MalE signal peptide operably
linked to a sequence coding for the GFP reporter protein.
[0021] FIG. 13 shows plasmid pAKLP2 comprising the L-rhamnose
inducible rhaBAD promoter and a sequence (nitA) coding for a
Nitrilase protein.
[0022] FIG. 14 shows SDS-PAGE of cells of the induced Pseudomonas
putida strain KT2440 (pAKLP2). Samples were taken after different
time intervals as indicated. An arrow indicates the Nitrilase
protein. M=Mark12, molecular weight standard of Invitrogen.
[0023] FIG. 15 shows plasmid pAKLP1 comprising the L-rhamnose
inducible rhaBAD promoter and sequences coding for the Fab-M heavy
and light chains which are operably linked to a sequence coding for
the OmpA signal peptide and a sequence coding for the PhoA signal
peptide, respectively.
[0024] FIG. 16 shows SDS-PAGE of cells of the induced Pseudomonas
putida strain KT2440 (pAKLP1). Samples were taken after different
time intervals as indicated. The arrows indicate the FabM heavy and
light chains. M=Mark12, molecular weight standard of
Invitrogen.
[0025] FIG. 17 shows SDS-PAGE of fermentation samples of the
Escherichia coli strain W3110 (pBW22-pelB-S1). Samples were taken
after different time intervals (in hours) as indicated. An arrow
indicates the scFv protein. M=Mark12, molecular weight standard of
Invitrogen.
DETAILED DESCRIPTION OF THE INVENTION
[0026] As used herein, the following definitions are supplied in
order to facilitate the understanding of the present invention.
[0027] A "vector expressible in a host" or "expression vector" is a
polynucleic acid construct, generated recombinantly or
synthetically, with a series of specified polynucleic acid elements
that permit transcription of a particular nucleic acid sequence in
a host cell. Typically, this vector includes a transcriptional unit
comprising a particular nucleic acid sequence to be transcribed
operably linked to a promoter. A vector expressible in a host can
be e. g. an autonomously or self-replicating plasmid, a cosmid, a
phage, a virus or a retrovirus.
[0028] The terms "host", "host cell" and "recombinant host cell"
are used interchangeably herein to indicate a prokaryotic cell into
which one or more vectors or isolated and purified nucleic acid
sequences of the invention have been introduced. It is understood
that such terms refer not only to the particular subject cell but
also to the progeny or potential progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0029] The term "comprise" is generally used in the sense of
include, that is to say permitting the presence of one or more
features or components.
[0030] "Promoter" as used herein refers to a nucleic acid sequence
that regulates expression of a transcriptional unit. A "promoter
region" is a regulatory region capable of binding RNA polymerase in
a cell and initiating transcription of a downstream (3' direction)
coding sequence. Within the promoter region will be found a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase such as
the putative -35 region and the Pribnow box.
[0031] "L-rhamnose operon" refers to the rhaSR-rhaBAD operon as
described for E. coli in Holcroft and Egan, 2000, J. Bacteriol. 182
(23), 6774-6782. The rhaBAD operon is a positively regulated
catabolic operon which transcribes RhaB, RhaA and RhaD divergently
from another rha operon, rhaSR, with approximately 240 bp of DNA
separating their respective transcription start sites. The rhaSR
operon encodes the two L-rhamnose-specific activators RhaS and
RhaR. RhaR regulates transcription of rhaSR, whereas RhaS bind DNA
upstream at -32 to -81 relative to the transcription start site of
rhaBAD. Furthermore the rhaSR-rhaBAD intergenic operon contains CRP
binding sites at positions -92,5 (CRP 1) relative to the
transcription start site of rhaBAD and CRP binding sites at
positions -92,5 (CRP 2), -115,5 (CRP 3) and 116,5 (CRP 4) relative
to the transcription start site of rhaSR as well as a binding site
for RhaR spanning -32 to -82 relative to the transcription start
site of rhaSR.
[0032] With "rhaBAD promoter region of the L-rhamnose operon" is
meant the rhaBAD operon consisting essentially of the rhaBAD
transcription initiation site, the putative -35 region, the Pribnow
box, the CRP binding site CPR1, the binding site for RhaS relative
to the transcription start site of rhaBAD as well as CRP binding
sites CRP 2-4, and binding site for RhaR relative to the
transcription start site of rhaSR. With "rhaBAD promoter" is meant
the promoter of the rhaBAD operon consisting essentially of the
rhaBAD transcription initiation site, the putative -35 region, the
Pribnow box, the binding site for RhaS and the CRP1 binding site
region relative to the transcription start site of rhaBAD, and the
CRP binding site CRP4 or a part thereof relative to the
transcription start site of rhaSR.
[0033] "CRP" means "Catabolite regulator protein". "CRP" is often
referred in the art as "cyclic AMP receptor protein", which has the
synonymous meaning. CRP is a regulator protein controlled by cyclic
AMP (cAMP) which mediates the activation of catabolic operons such
as the L-rhamnose operon.
[0034] An "enhancer" is a nucleic acid sequence that acts to
potentiate the transcription of a transcriptional unit independent
of the identity of the transcriptional unit, the position of the
sequence in relation to the transcriptional unit, or the
orientation of the sequence. The vectors of the present invention
optionally include enhancers.
[0035] "Transcriptional unit" as used herein refers to a nucleic
acid sequence that is normally transcribed into a single RNA
molecule. The transcriptional unit might contain one gene
(monocistronic) or two (dicistronic) or more genes (polycistronic)
that code for functionally related polypetide molecules.
[0036] A nucleic acid sequence is "operably linked" when it is
placed into a functional relationship with another nucleic acid
sequence. For example, DNA for a signal sequence is operably linked
to DNA for a protein if it is expressed as a preprotein that
participates in the secretion of the protein; a promoter is
operably linked to a coding sequence if it affects the
transcription of the sequence; or a translation initiation region
such as a ribosome binding site is operably linked to a nucleic
acid sequence encoding e. g. a polypeptide if it is positioned so
as to facilitate translation of the polypeptide. Linking can be
accomplished by ligation at convenient restriction sites. If such
sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
[0037] "Nucleic acid" or "nucleic acid sequence" or "isolated and
purified nucleic acid or nucleic acid sequence" as referred in the
present invention might be DNA, RNA, or DNA/RNA hybrid. In case the
nucleic acid or the nucleic acid sequence is located on a vector it
is usually DNA. DNA which is referred to herein can be any
polydeoxynuclotide sequence, including, e.g. double-stranded DNA,
single-stranded DNA, double-stranded DNA wherein one or both
strands are composed of two or more fragments, double-stranded DNA
wherein one or both strands have an uninterrupted phosphodiester
backbone, DNA containing one or more single-stranded portion(s) and
one or more double-stranded portion(s), double-stranded DNA wherein
the DNA strands are fully complementary, double-stranded DNA
wherein the DNA strands are only partially complementary, circular
DNA, covalently-closed DNA, linear DNA, covalently cross-linked
DNA, cDNA, chemically-synthesized DNA, semi-synthetic DNA,
biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA,
sheared DNA, labeled DNA, such as radiolabeled DNA and
fluorochrome-labeled DNA, DNA containing one or more non-naturally
occurring species of nucleic acid. DNA sequences can be synthesized
by standard chemical techniques, for example, the phosphotriester
method or via automated synthesis methods and PCR methods. The
purified and isolated DNA sequence may also be produced by
enzymatic techniques.
[0038] RNA which is referred to herein can be e.g. single-stranded
RNA, cRNA, double-stranded RNA, double-stranded RNA wherein one or
both strands are composed of two or more fragments, double-stranded
RNA wherein one or both strands have an uninterrupted
phosphodiester backbone, RNA containing one or more single-stranded
portion(s) and one or more double-stranded portion(s),
double-stranded RNA wherein the RNA strands are fully
complementary, double-stranded RNA wherein the RNA strands are only
partially complementary, covalently crosslinked RNA,
enzyme-digested RNA, sheared RNA, mRNA, chemically-synthesized RNA,
semi-synthetic RNA, biosynthetic RNA, naturally-isolated RNA,
labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA,
RNA containing one or more non-naturally-occurring species of
nucleic acid.
[0039] With "variants" or "variants of a sequence" is meant a
nucleic acid sequence that vary from the reference sequence by
conservative nucleic acid substitutions, whereby one or more
nucleic acids are substituted by another with same characteristics.
Variants encompass as well degenerated sequences, sequences with
deletions and insertions, as long as such modified sequences
exhibit the same function (functionally equivalent) as the
reference sequence.
[0040] As used herein, the terms "polypeptide", "peptide",
"protein", "polypeptidic" and "peptidic" are used interchangeably
to designate a series of amino acid residues connected to the other
by peptide bonds between the alpha-amino and carboxy groups of
adjacent residues.
[0041] The term "isolated and purified nucleic acid sequence"
refers to the state in which the nucleic acid sequence will be, in
accordance with the present invention. The nucleic acid sequence
will be free or substantially free of material with which they are
naturally associated such as other nucleic acids with which they
are found in their natural environment, or the environment in which
they are prepared (e. g. cell culture) when such preparation is by
recombinant technology practised in vitro or in vivo.
[0042] The terms "transformation", "transformed" or "introducing a
nucleic acid into a host cell" denote any process wherein an
extracellular nucleic acid like a vector, with or without
accompanying material, enters a host cell. The term "cell
transformed" or "transformed cell" means the cell or its progeny
into which the extracellular nucleic acid has been introduced and
thus harbours the extracellular nucleic acid. The nucleic acid
might be introduced into the cell so that the nucleic acid is
replicable either as a chromosomal integrant or as an extra
chromosomal element. Transformation of appropriate host cells with
e. g. an expression vector can be accomplished by well known
methods such as microinjection, electroporation, particle
bombardement or by chemical methods such as Calcium
phosphate-mediated transformation, described e. g. in Maniatis et
al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring
Harbor Laboratory or in Ausubel et al. 1994, Current protocols in
molecular biology, John Wiley and Sons.
[0043] "Heterologous nucleic acid sequence" or "nucleic acid
sequence heterologous to a host" means a nucleic acid sequence
which encodes e. g. an expression product such as a polypeptide
that is foreign to the host ("heterologous expression" or
"heterologous product") i. e. a nucleic acid sequence originating
from a donor different from the host or a chemically synthesized
nucleic acid sequence which encodes e. g. an expression product
such as a polypeptide that is foreign to the host. In case the host
is a particular prokaryotic species, the heterologous nucleic acid
sequence is preferably originated from a different genus or family,
more preferred from a different order or class, in particular from
a different phylum (division) and most particular from a different
domain (empire) of organisms.
[0044] The heterologous nucleic acid sequence originating from a
donor different from the host can be modified, before it is
introduced into a host cell, by mutations, insertions, deletions or
substitutions of single nucleic acids or a part of the heterologous
nucleic acid sequence as long as such modified sequences exhibit
the same function (functionally equivalent) as the reference
sequence. A heterologous nucleic acid sequence as referred herein
encompasses as well nucleic sequences originating from a different
domain (empire) of organisms such as from eukaryotes (of eukaryotic
origin) such as e. g. human antibodies which have been used in
phage display libraries and of which single nucleic acids or a part
of the nucleic acid sequences have been modified according to the
"codon usage" of a prokaryotic host.
[0045] "Signal sequence" or "signal peptide sequence" refers to a
nucleic acid sequence which encodes a short amino acid sequence
(i.e., signal peptide) present at the NH2-terminus of certain
proteins that are normally exported by cells to non-cytoplasmic
locations (e.g., secretion) or to be membrane components. Signal
peptides direct the transport of proteins from the cytoplasm to
non-cytoplasmic locations.
[0046] "Translation initiation region" is a signal region which
promotes translation initiation and which functions as the ribosome
binding site such as the Shine Dalgarno sequence.
[0047] "Transcription termination region" refers to a sequence
which causes RNA polymerase to terminate transcription. The
transcription termination region is usually part of a
transcriptional unit and increases the stability of the mRNA.
[0048] "Antibody" refers to a class of plasma proteins produced by
the B-cells of the immune system after stimulation by an antigen.
Mammal (i.e. Human) antibodies are immunoglobulins of the Ig G, M,
A, E or D class. The term "antibody" as used for the purposes of
this invention includes, but is not limited to, polyclonal
antibodies, monoclonal antibodies, anti-idiotypic antibodies and
auto-antibodies present in autoimmune diseases, such as diabetes,
multiple sclerosis and rheumatoid arthritis as well as chimeric
antibodies. The basic antibody structural unit is known to comprise
a tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function.
[0049] Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, and
define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 more amino acids.
[0050] The term antibody is used to mean whole antibodies and
binding fragments thereof. Binding fragments include single chain
fragments, Fv fragments and Fab fragments.
[0051] The term "single-chain antibody" includes such non-natural
antibody formats which combine only the antigen-binding regions of
antibodies on a single stably-folded polypeptide chain. As such,
single-chain antibodies are of considerably smaller size than
classical immunoglobulins but retain the antigen-specific binding
properties of antibodies. Single-chain antibodies are widely used
for a variety of different applications, including for example as
therapeutics, diagnostics, research tools etc.
[0052] The term Fab fragment is sometimes used in the art to mean
the binding fragment resulting from papain cleavage of an intact
antibody. The terms Fab' and F(ab')2 are sometimes used in the art
to refer to binding fragments of intact antibodies generated by
pepsin cleavage. In the context of the present invention, Fab is
used to refer generically to double chain binding fragments of
intact antibodies having at least substantially complete light and
heavy chain variable domains sufficient for antigen-specific
bindings, and parts of the light and heavy chain constant regions
sufficient to maintain association of the light and heavy chains.
An example of such Fab is described in Skerra et al., 1988, Science
240(4855), 1038-41, for instance. A Fab fragment e. g. of the IgG
idiotype might or might not contain at least one of the two
cysteine residues that form the two inter-chain disulfide bonds
between the two heavy chains in the intact immunoglobulin. Usually,
Fab fragments are formed by complexing a full-length or
substantially full-length light chain with a heavy chain comprising
the variable domain and at least the CH1 domain of the constant
region. In addition, the C-terminal cysteine on the light chain may
be replaced with serine or another amino acid to eliminate the
interchain disulfide bond between the heavy and light chains
according to the present invention.
[0053] Further encompassed are chimeric antibodies which are
antibodies whose light and heavy chain genes have been constructed,
typically by genetic engineering, from immunoglobulin gene segments
(e. g., segments encoding the variable region and segments encoding
the constant region), for example, belonging to different species.
For example, the variable (V) segments of the genes from a mouse
monoclonal antibody can be joined to human constant (C) segments,
such as IgG1 an IgG4. A typical chimeric antibody is thus a hybrid
protein consisting of the V or antigen-binding domain from a mouse
antibody and a C or effector domain from a human antibody. Chimeric
antibodies have the same or similar binding specificity and
affinity as a mouse or other nonhuman antibody that provides the
variable regions of the antibody.
[0054] The term "human antibody" includes antibodies having
variable and constant regions (if present) derived from human
germline immunoglobulin sequences including either natural or
artificial, engineered affinity maturation. Human antibodies of the
invention can include amino acid residues not encoded by human
germline immunoglobulin sequences (e. g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). However, the term "human antibody" does include
antibodies in which CDR sequences derived from the germline of
another mammalian species, such as a mouse, have been grafted onto
human framework sequences (i. e. humanized antibodies). Functional
variants of such "human antibodies", e.g. truncated versions
thereof or engineered muteins where e.g. individual proline or
cysteine residues have been engineered by the means of genetic
engineering well known in the art are encompassed by the term, in
contrast. Examples of such may be found e.g. in WO 98/02462.
However, the term only relates to the amino acid sequence of such
antibody, irrespective of any glycosylation or other chemical
modification of the peptide backbone.
[0055] In one aspect, the present invention provides a vector
expressible in a host comprising the rhaBAD promoter region of the
L-rhamnose operon operably linked to a transcriptional unit
comprising [0056] a) a nucleic acid sequence which is heterologous
to said host [0057] b) a prokaryotic signal sequence operably
linked to said nucleic acid sequence, whereas said prokaryotic
signal sequence is selected from signal peptides of periplasmatic
binding proteins for sugars, amino acids, vitamins and ions and,
whereas the expression of said nucleic acid sequence is controlled
by said promoter region.
[0058] The vector according to the invention is preferably an
autonomously or self-replicating plasmid, a cosmid, a phage, a
virus or a retrovirus. A wide variety of host/vector combinations
may be employed in expressing the nucleic acid sequences of this
invention. Useful expression vectors, for example, may consist of
segments of chromosomal, non-chromosomal and/or synthetic nucleic
acid sequences. Suitable vectors include vectors with specific host
range such as vectors specific for e. g. E. coli as well as vectors
with broad-host-range such as vectors useful for Gram-negative
bacteria. "Low-copy", "medium-copy" as well as "high copy" plasmids
can be used.
[0059] Useful vectors for e. g. expression in E. coli are: pQE70,
pQE60 und pQE-9 (QIAGEN, Inc.); pBluescript Vektoren, Phagescript
Vektoren, pNH8A,pNH16a,pNH18A, pNH46A (Stratagene Cloning Systems,
Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia
Bio-tech, Inc.); pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30,
pRep4, pACYC177, pACYC184, pRSF1010 and pBW22 (Wilms et al., 2001,
Biotechnology and Bioengineering, 73 (2) 95-103) or derivates
thereof such as plasmid pBW22-Fab-H or plasmid pAKL14. Further
useful plasmids are well known to the person skilled in the art and
are described e.g. in "Cloning Vectors" (Eds. Pouwels P. H. et al.
Elsevier, Amsterdam-New York-Oxford, 1985).
[0060] Preferred vectors of the present inventions are autonomously
or self-replicating plasmids, more preferred are vectors with
specific host range such as vectors specific for e. g. E. coli.
Most preferred are pBR322, pUC18, pACYC177, pACYC184, pRSF1010 and
pBW22 or derivates thereof such as pBW22-Fab-H or pAKL14, in
particular pBW22-Fab-H or pAKL14, most particular pAKL14.
[0061] In a preferred embodiment, the rhaBAD promoter region of the
L-rhamnose operon is the rhaBAD promoter. In a particular preferred
embodiment, the rhaBAD promoter consists of the sequence SEQ ID NO.
1, a sequence complementary thereof and variants thereof.
Preferably the rhaBAD promoter region of the L-rhamnose operon, the
rhaBAD promoter and the rhaBAD promoter consisting of the sequence
SEQ ID NO. 1, a sequence complementary thereof and variants thereof
are from the L-rhamnose operon of E. coli.
[0062] In another preferred embodiment of the invention the vector
expressible in a prokaryotic host comprises apart from the rhaBAD
promoter region of the L-rhamnose operon operably linked to a
transcriptional unit furthermore sequences encoding the
L-rhamnose-specific activators RhaS and RhaR including their
respective native promoter sequences. Upon expression the RhaS and
RhaR proteins control the activity of the rhaBAD promoter.
[0063] As prokaryotic signal sequence selected from signal peptides
of periplasmatic binding proteins for sugars, amino acids, vitamins
and ions, signal peptides such as PelB (Erwinia chrysantemi,
Pectate lyase precursor), PelB (Erwinia carotovora, Pectate lyase
precursor), PelB (Xanthomonas campestris, Pectate lyase precursor),
LamB (E. coli, Maltoporin precursor), MalE (E. coli,
Maltose-binding protein precursor), Bla (E. coli, Beta-lactamase),
OppA (E. coli, Periplasmic oligopeptide-binding protein), TreA (E.
coli, periplasmic trehalase precursor), MppA (E. coli, Periplasmic
murein peptide-binding protein precursor), BglX (E. coli,
Periplasmic beta-glucosidase precursor), ArgT (E. coli,
Lysine-arginine-ornithine binding periplasmic protein precursor),
MalS (E. coli, Alpha-amylase precursor), HisJ (E. coli,
Histidine-binding periplasmic protein precursor), XylF (E. coli,
D-Xylose-binding periplasmic protein precursor), FecB (E. coli,
dicitrate-binding periplasmic protein precursor), OmpA (E. coli,
outer membrane protein A precursor) and PhoA (E. coli, Alkaline
phosphatase precursor) can be used.
[0064] In a preferred embodiment, the signal sequence is selected
from the E. coli signal peptides LamB (Maltoporin precursor), MalE
(Maltose-binding protein precursor), Bla (Beta-lactamase), OppA
(Periplasmic oligopeptide-binding protein), TreA (periplasmic
trehalase precursor), MppA (Periplasmic murein peptide-binding
protein precursor), BglX (Periplasmic beta-glucosidase precursor),
ArgT (Lysine-arginine-ornithine binding periplasmic protein
precursor), MalS (Alpha-amylase precursor), HisJ (Histidine-binding
periplasmic protein precursor), XylF (D-Xylose-binding periplasmic
protein precursor), FecB (dicitrate-binding periplasmic protein
precursor), OmpA (outer membrane protein A precursor) and PhoA
(Alkaline phosphatase precursor). These are particularly useful for
heterologous expression in E. coli. More preferred are the E. Coli
signal peptides LamB (Maltoporin precursor), MalE (Maltose-binding
protein precursor), Bla (Beta-lactamase), TreA (periplasmic
trehalase precursor), ArgT (Lysine-arginine-ornithine binding
periplasmic protein precursor), FecB (dicitrate-binding periplasmic
protein precursor). Most particular preferred are the E. coli
signal peptides LamB (Maltoporin precursor) and MalE
(Maltose-binding protein precursor). In case a dicistronic or
polycistronic transcriptional unit is used, different or identical
signal sequences operably linked to each of the cistrons can be
applied. Preferably different signal sequences are used in such a
case. The signal sequences to be employed in the expression vectors
of the present invention can be obtained commercially or
synthesized chemically. For example, signal sequences can be
synthesized according to the solid phase phosphoramidite triester
method described, e.g., in Beaucage & Caruthers, Tetrahedron
Letts. 22:1859-1862 (1981), using an automated synthesizer, as
described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168
(1984). Purification of oligonucleotides can be performed by either
native acrylamide gel electrophoresis or by anion-exchange HPLC as
described in Pearson & Reanier, J. Chrom. 255:137-149
(1983).
[0065] The transcriptional unit according to the present invention
usually further comprises a translation initiation region upstream
of the initiation point of the translation of said transcriptional
unit, said translation initiation region consisting of the sequence
AGGAGATATACAT (SEQ ID NO. 2), whereas said translation initiation
region is operably linked to said nucleic acid sequence. The
sequence AGGAGATATACAT (SEQ ID NO. 2) is usually located upstream
directly adjacent to the initiation point of the translation of the
transcriptional unit which can be ATG, GTG or TTG.
[0066] Usually, said transcriptional unit further comprises a
transcription termination region selected from rrnB, RNA I, T7Te,
rrnB T1, trp a L126, trp a, tR2, T3Te, P14, tonB t, and trp a L153.
Preferably, the rrnB transcriptional terminator sequence is
used.
[0067] The heterologous nucleic acid sequence according to the
present invention encodes an expression product that is foreign to
the host. In case the host is a prokaryotic species such as E. coli
the nucleic acid sequence of interest is more preferably from
another class like the gammaproteobacteria such as from e.g.
Burkholderia sp., in particular from a different phylum such as
archae bacteria, and most particular from an eukaryotic organism
such as mammals in particular from humans. However, the
heterologous nucleic acid sequence might be modified according to
the "codon usage" of the host. The heterologous sequence according
to the present invention is usually a gene of interest. The gene of
interest preferably encodes a heterologous polypeptide such as a
structural, regulatory or therapeutic protein, or N-- or C-terminal
fusions of structural, regulatory or therapeutic protein with other
proteins ("Tags") such as green fluorescent protein or other fusion
proteins. The heterologous nucleic acid sequence might encode as
well a transcript which can be used in the form of RNA, such as e.
g. antisense-RNA.
[0068] The protein may be produced as an insoluble aggregate or as
a soluble protein which is present in the cytoplasm or in the
periplasmic space of the host cell, and/or in the extracellular
medium. Preferably, the protein is produced as a soluble protein
which is present in the periplasmic space of the host cell and/or
in the extracellular medium. Examples of proteins include hormones
such as growth hormone, growth factors such as epidermal growth
factor, analgesic substances like enkephalin, enzymes like
chymotrypsin, antibodies, receptors to hormones and includes as
well proteins usually used as a visualizing marker e.g. green
fluorescent protein.
[0069] Other proteins of interest are growth factor receptors
(e.g., FGFR, PDGFR, EFG, NGFR, and VEGF) and their ligands. Other
proteins are G-protein receptors and include substance K receptor,
the angiotensin receptor, the [alpha]- and [beta]-adrenergic
receptors, the serotonin receptors, and PAF receptor (see, e.g.
Gilman, Ann. Rev. Biochem. 56, 625-649 (1987). Other proteins
include ion channels (e.g., calcium, sodium, potassium channels),
muscarinic receptors, acetylcholine receptors, GABA receptors,
glutamate receptors, and dopamine receptors (see Harpold, U.S. Pat.
Nos. 5,401,629 and 5,436,128). Other proteins of interest are
adhesion proteins such as integrins, selecting, and immunoglobulin
superfamily members (see Springer, Nature 346, 425-433 (1990).
Osborn, Cell 62, 3 (1990); Hynes, Cell 69, 11 (1992)). Other
proteins are cytokines, such as interleukins IL-1 through IL-13,
tumor necrosis factors [alpha] and [beta], interferons [alpha],
[beta], and [gamma], tumor growth factor Beta (TGF-[beta]), colony
stimulating factor (CSF) and granulocyte monocyte colony
stimulating factor (GM-CSF) (see Human Cytokines: Handbook for
Basic & Clinical Research. Aggrawal et al. eds., Blackwell
Scientific, Boston, Mass. 1991). Other proteins of interest are
intracellular and intercellular messengers, such as, adenyl
cyclase, guanyl cyclase, and phospholipase C. Drugs are also
proteins of interest. The heterologous protein of interest can be
of human, mammalian or prokaryotic origin. Other proteins are
antigens, such as glycoproteins and carbohydrates from microbial
pathogens, both viral and bacterial, and tumors. Other proteins are
enzymes like chymosin, proteases, polymerases, dehydrogenases,
nucleases, glucanases, oxidases, .alpha.-amylase, oxidoreductases,
lipases, amidases, nitril hydratases, esterases or nitrilases.
[0070] Preferably, the heterologous nucleic acid sequence,
according to the present invention, encodes a polypeptide, more
preferably an antibody and most preferably a Fab fragment. In
particular a human antibody or a humanised antibody, more
particularly a human Fab fragment is encoded by the nucleic acid
sequence. The human Fab fragment encoded by the nucleic acid
sequence is preferably either a human antibody fragment or a human
antibody fragment that was grafted with at least one CDR from
another mammalian species.
[0071] In one more preferred embodiment, the human Fab fragment is
a fully human HuCAL-Fab as obtainable from an artificial,
consensus-framework-based human antibody phage library that was
artifically randomized in the CDR as described by Knappik et al.,
2000, J. Mol. Biol. 296 (1), 57-86.
[0072] In another more preferred optional embodiment, the,
optionally chimeric, CDR grafted, human Fab fragment is a
non-HuCAL-Fab as opposed to the HuCAL-Fab definition in the
foregoing, which in case of a fully human Fab fragment preferably
means that it does not share the HuCAL consensus sequence framework
but its non-CDR sequence portions are at least 70% more preferably
85%, most preferably 95% identical in amino acid sequence to the
respective variable and constant light and heavy chains
germline-encoded sequences, additionally and more preferably that
its CDRs are directly obtained from naturally occurring genomic
sequences of lymphoid cells including genomic affinity maturation
events.
[0073] The Fab fragment is preferably derived from an IgG antibody
and does not contain cysteine residues that form the two interchain
disulfide bonds between the two heavy chains in the intact
immunoglobulin. In particular, the heavy and the light chain of the
antibody or preferably of the Fab fragment are encoded by a
dicistronic transcriptional unit, whereas each chain is operably
linked to a prokaryotic signal sequence selected from signal
peptides of periplasmatic binding proteins for sugars, amino acids,
vitamins and ions and an identical translation initiation region
upstream of the initiation point of the translation of the
transcriptional unit. Preferably, the translation initiation region
consists of the sequence AGGAGATATACAT (SEQ ID NO. 2).
[0074] In the present invention, the order and the distance in
which the signal sequence and the heterologous nucleic acid
sequence are arranged within the expression vectors can be varied.
In preferred embodiments, the signal sequence is 5' (upstream) to
the nucleic acid sequence encoding e. g. the polypeptide of
interest. The signal peptide sequence and the nucleic acid sequence
encoding e. g. the polypeptide of interest can be separated by zero
to about 1000 amino acids. In preferred embodiments, the signal
peptide sequence and nucleic acid sequence encoding e. g. the
polypeptide of interest are directly adjacent to each other, i.e.
separated by zero nucleic acids.
[0075] Preferably, the rhaBAD promoter region and the operably
linked transcriptional unit of the vector of the present invention
consists of the sequence SEQ ID NO. 3, a sequence complementary
thereof and variants thereof.
[0076] More preferably, the rhaBAD promoter region and the operably
linked transcriptional unit of the vector of the present invention
consist of the sequence SEQ ID NO. 4, a sequence complementary
thereof and variants thereof.
[0077] Also encompassed by the present invention is the use of a
vector according to the invention for the regulated heterologous
expression of a nucleic acid sequence in a prokaryotic host. The
expression can be regulated by the amount of L-rhamnose available
to the prokaryotic host. Usually, the amount of L-rhamnose in the
medium of the cultured prokaryotic host is between 0.01 and 100
g/l, preferably between 0.1 and 10 g/l, more preferably between 1
and 5 g/l.
[0078] Preferably, the heterologous nucleic acid sequence encodes
for a polypeptide, more preferably for an antibody and most
preferably for a Fab fragment, whereas the heavy and light chains
of the antibody or the Fab fragment are expressed in equal amounts,
thus leading to high concentrations of functional antibody or Fab
fragment. In particular a human antibody or a humanised antibody
more particular a human Fab fragment, most particular a human Fab
fragment as described above is encoded by the heterologous nucleic
acid sequence.
[0079] In order to obtain high concentrations of functional
antibody or Fab fragment it is essential to have an equal amount of
the heavy and light chains being expressed. In case one of both
chains is overproduced compared to the other chain, non-reducible
high molecular weight immunoreactive aggregates can be built, which
is undesirably. It has been surprisingly found that with the
vectors of the present invention high titers of functional
antibodies can be obtained whereas only very low amounts of
overproduced light or heavy chain or high molecular weight
immunoreactive aggregates are built. Usually, less than 20%,
preferably less than 10% of the expressed amount of antibody or Fab
fragment are expressed as overproduced light or heavy chain or high
molecular weight immunoreactive aggregates. The amount of the heavy
and light chains overproduced and of high molecular weight
immunoreactive aggregates can be measured by analysing extracts of
the host expressing the antibody or the Fab fragment such as
lysozyme extracts of the cultured host cell using SDS-PAGE or
Western blot.
[0080] In still another aspect, the invention provides an isolated
and purified nucleic acid sequence expressible in a host comprising
the rhaBAD promoter region of the L-rhamnose operon operably linked
to a transcriptional unit comprising [0081] a) a nucleic acid
sequence which is heterologous to said host [0082] b) a prokaryotic
signal sequence operably linked to said nucleic acid sequence,
whereas said prokaryotic signal sequence is selected from signal
peptides of periplasmatic binding proteins for sugars, amino acids,
vitamins and ions and, whereas the expression of said nucleic acid
sequence is controlled by said promoter region. The rhaBAD promoter
is the preferred promoter region. More preferred, the isolated and
purified nucleic acid sequence consists of SEQ ID NO. 1, a sequence
complementary thereof and variants thereof, in particular the
isolated and purified nucleic acid sequence consists of SEQ ID NO.
3, a sequence complementary thereof and variants thereof, most
particular the isolated and purified nucleic acid sequence consists
of SEQ ID NO. 4, a sequence complementary thereof and variants
thereof.
[0083] The isolated and purified nucleic acid sequence of this
invention can be isolated according to standard PCR protocols and
methods well known in the art. Said purified and isolated DNA
sequence can further comprise one or more regulatory sequences, as
known in the art e.g. an enhancer, usually employed for the
expression of the product encoded by the nucleic acid sequence.
[0084] In order to select host cells successfully and stably
transformed with the vector or the isolated and purified nucleic
acid sequence of the present invention, a gene that encodes a
selectable marker (e. g., resistance to antibiotics) can be
introduced into the host cells along with the nucleic acid sequence
of interest. The gene that encodes a selectable marker might be
located on the vector or on the isolated and purified nucleic acid
sequence or might optionally be co-introduced in separate form e.g.
on a separate vector. Various selectable markers can be used
including those that confer resistance to antibiotics, such as
hygromycin, ampicillin and tetracyclin. The amount of the
antibiotic can be adapted as desired in order to create selective
conditions. Usually, one selectable marker is used. As well
reporter genes such as fluorescent proteins can be introduced into
the host cells along with the nucleic acid sequence of interest, in
order to determine the efficiency of transformation.
[0085] Another aspect of the present invention is to provide a
prokaryotic host transformed with a vector of the present
invention. In a particular embodiment of the invention the
prokaryotic host is transformed with plasmid pBW22-Fab-H or plasmid
pAKL14, preferably with plasmid pAKL14 comprising two different
coding regions in its dicistronic expression cassette for
expressing a secreted, heterodimeric protein in such host cell such
as e.g. a Fab. Preferably such heterodimeric protein is a Fab. In
another embodiment of the invention the prokaryotic host is
transformed with the isolated and purified nucleic acid sequence of
the present invention.
[0086] A wide variety of prokaryotic host cells can be used for the
heterologous expression of the nucleic acid sequences of this
invention. These hosts may include strains of Gram-negative cells
such as E. coli and Pseudomonas, or Gram postitive cells such as
Bacillus and Streptomyces. Preferably, the host cell is a
Gram-negative cell, more preferably an E. coli cell. E. coli which
can be used are e. g. the strains TG1, W3110, DH1, XL1-Blue and
Origami, which are commercially available or can be obtained via
the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH, Braunschweig, Germany). Most preferably, W3110 is used. The
host cell might or might not metabolise L-rhamnose. A host cell
which is ordinarily capable to uptake and metabolise L-rhamnose
like E. coli might be modified to be deficient in one or more
functions related to the uptake and/or metabolism of L-rhamnose.
Deficiency in one or more functions related to the uptake and/or
metabolism of L-rhamnose can be achieved by e.g. suppressing or
blocking the expression of a gene coding for a protein related to
the uptake and/or metabolism of L-rhamnose such as the gene rhaB
coding for L-rhamnulose kinase. This can be done by known
techniques such as transposon supported mutagenesis or knock-out
mutation. Usually, the prokaryotic host corresponds to the signal
sequences chosen, e. g. in case signal sequences of E. coli are
used, the host cell is usually a member of the same family of the
enterobacteriaceae, more preferably the host cell is an E. coli
strain.
[0087] Further provided with the present invention is a method for
producing a polypeptide in a host cell, comprising the steps of
[0088] a) constructing a vector, [0089] b) transforming a
prokaryotic host with said vector, [0090] c) allowing expression of
said polypetide in a cell culture system under suitable conditions,
[0091] d) recovering said polypeptide from the cell culture
system.
[0092] The vector used, as well as its construction and the
transformation of a prokaryotic host are as defined above, whereas
the heterologous nucleic acid sequence comprised by the vector
encodes a polypeptide. Preferably, the polypeptide produced is an
antibody and most preferably a Fab fragment, whereas the heavy and
light chains of the antibody or the Fab fragment are expressed in
the cell culture system in equal amounts, thus leading to high
concentrations of functional antibody or Fab fragment.
[0093] As cell culture system continuous or discontinous culture
such as batch culture or fed batch culture can be applied in
culture tubes, shake flasks or bacterial fermentors. Host cells are
usually cultured in conventional media as known in the art such as
complex media like "nutrient yeast broth medium" or a glycerol
containing medium as described by Kortz et al., 1995, J.
Biotechnol. 39, 59-65 or a mineral salt media as described by Kulla
et al., 1983, Arch. Microbiol, 135, 1. The preferred medium for
carrying out the expression of said polypeptide is a glycerol
containing medium, more preferably the medium described by Kortz et
al., 1995, J. Biotechnol. 39, 59-65.
[0094] The medium might be modified as appropriate e.g. by adding
further ingredients such as buffers, salts, vitamins or amino
acids. As well different media or combinations of media can be used
during the culturing of the cells. Preferably, the medium used as
basic medium should not include L-rhamnose, in order to achieve a
tight regulation of the L-rhamnose promoter region. L-rhamnose is
usually added after the culture has reached an appropriate
OD.sub.600 depending on the culture system. Usually, the amount of
L-rhamnose in the medium of the cultured prokaryotic host is
between 0.01 and 100 g/l, preferably between 0.1 and 10 g/l, more
preferably 1 and 5 g/l. For batch culture the usual OD.sub.600 is
usually 0.4 or higher. Appropriate pH ranges are e. g. 6-8
preferably 7-7.5, appropriate culture temperatures are between 10
and 40, preferably between 20 and 37.degree. C. The cells are
incubated usually as long as it takes until the maximum amount of
expressed product has accumulated, preferably between 1 hour and 20
days, more preferably between 5 hours and 3 days. The amount of
expressed product depends on the culture system used. In shake
flask culture usually expressed product in the amount of 0.5 g/l
culture medium can be produced with a host transformed with the
vector of the present invention. Using a fermentor culture in a
batch and/or fed-batch mode expressed product in the amount of
usually more than 0.5 g/l fermentation broth, preferably more than
1 g/l, more preferably more than 1.3 g/l can be obtained.
[0095] Following expression in the host cell, the expressed product
such as the polypeptide of interest can then be recovered from the
culture of host cells. When the polypeptide of interest are
immunoglobulin chains, the heavy chain and the light chain are
normally each expressed in the host cell and secreted to the
periplasm of the cell. The signal peptides encoded by the signal
sequences in the expression vector are then processed from the
immunoglobulin chains. The mature heavy and light chains are then
assembled to form an intact antibody or a Fab fragment. In order to
obtain a maximum yield of the expressed product the cells are
usually harvested at the end of the culture and lysed, such as
lysing by lysozyme treatment, sonication or French Press. Thus, the
polypeptides are usually first obtained as crude lysate of the host
cells. They can then be purified by standard protein purification
procedures known in the art which may include differential
precipitation, molecular sieve chromatography, ion-exchange
chromatography, isoelectric focusing, gel electrophoresis,
affinity, and immunoaffinity chromatography. These well known and
routinely practiced methods are described in, e.g., Ausubel et al.,
supra., and Wu et al. (eds.), Academic Press Inc., N.Y.;
Immunochemical Methods In Cell And Molecular Biology. For example,
for purification of recombinantly produced immunoglobulins or Fab
fragments, they can be purified with immunoaffinity chromatography
by passage through a column containing a resin which has bound
thereto target molecules to which the expressed immunoglobulins can
specifically bind.
[0096] A further aspect of the present invention is a vector
expressible in a host comprising a promoter region operably linked
to a transcriptional unit comprising [0097] a) a nucleic acid
sequence which is heterologous to said host [0098] b) a translation
initiation region upstream of the initiation point of the
translation of said transcriptional unit, said translation
initiation region consisting of the sequence AGGAGATATACAT (SEQ ID
NO. 2),
[0099] whereas said translation initiation region is operably
linked to said nucleic acid sequence and the expression of said
nucleic acid sequence is controlled by said promoter region. The
promoter region might be an inducible or non-inducible promoter
region. Usually, an inducible promoter region of a catabolic operon
is used. As inducible promoter region of a catabolic operon
negatively regulated promoter systems such as the lactose [lac]
(Yanisch-Perron et al., 1985, Gene 33, 103-109), and the tryptophan
[trp] (Goeddel et al., 1980, Nature (London) 287, 411-416)
promoters, and the hybrid promoters derived from these two [tac and
trc] (Brosius, 1984,Gene 27 :161-172 ; Amann and Brosius, 1985,
Gene 40,183-190) as well as positively regulated promoter systems
such as the araB promoter inducible by Arabinose (WO 86 04356), the
rhamnose promoter rhaSB inducible by rhamnose (WO 03068956) or the
"rhaBAD promoter region of the L-rhamnose operon" of the present
invention can be used. Preferably, positively regulated catabolic
operons are used, more preferred is the "rhaBAD promoter region of
the L-rhamnose operon" of the present invention. As well functional
equivalents of these promoters which might be from various
prokaryotic organisms might be used. Functional equivalents are in
the case of positively regulated catabolic operons equivalents
which in the presence of inducer show increased expression activity
compared to their activity in the absence of inducer. The
expression activity in the presence of inducer is usually at least
two times, preferably at least five times, more preferably at least
ten times higher than in the absence of the inducer.
[0100] Usually, the vector further comprises a signal sequence
operably linked to said nucleic acid sequence. The signal sequence
can be prokaryotic or eukaryotic. Preferably prokaryotic signal
sequences are used. A prokaryotic signal sequence is preferably
selected from signal peptides of periplasmatic binding proteins for
sugars, amino acids, vitamins and ions as described above or from
other prokaryotic signal sequence known to the person in the art.
More preferably the prokaryotic signal sequence is selected from
signal peptides of periplasmatic binding proteins for sugars, amino
acids, vitamins and ions which are described above. Usually, the
nucleic acid sequence encodes a polypeptide, preferably an
antibody, more preferably a Fab fragment as described above.
[0101] In a particular embodiment, in case the nucleic acid
sequence encodes an antibody, preferably a Fab fragment, the heavy
and the light chain of the antibody, preferably of the Fab fragment
are encoded by a dicistronic transcriptional unit, whereas each
chain is operably linked to a signal sequence and the translation
initiation region consisting of the sequence AGGAGATATACAT (SEQ ID
NO. 2).
[0102] In a further aspect the present invention provides a method
for producing a polypeptide in a host, comprising the steps of:
[0103] a) constructing a vector, [0104] b) transforming a
prokaryotic host with said vector, [0105] c) allowing expression of
said polypeptide in a cell culture system under suitable
conditions, [0106] d) recovering said polypeptide from the cell
culture system.
[0107] Useful vectors and hosts are as described above. The
construction of the vector, the transformation of a prokaryotic
host and the cell culture can be conducted as described above,
whereas the heterologous nucleic acid sequence comprised by the
vector encodes a polypeptide. In case the polypeptide produced is a
Fab fragment, the heavy and light chains of the Fab fragment are
expressed in said cell culture system in equal amounts.
[0108] The present invention also relates to methods and means for
the intracellular heterologous expression of nucleic acids encoding
e.g. polypeptides in a prokaryotic host. In particular the present
invention relates to vectors for the intracellular expression of a
heterologous polypeptide in a prokaryotic host, whereby the vector
is expressible in a prokaryotic host comprising the rhaBAD promoter
region of the L-rhamnose operon operably linked to a
transcriptional unit comprising a nucleic acid sequence which is
heterologous to said host. Since in this embodiment of the vector
of the present invention the nucleic acid sequence is not linked to
a prokaryotic signal sequence upon transforming a prokaryotic host
cell with the vector and expression of the polypeptide encoded by
the heterologous nucleic acid the polypeptide will not be
transported from the cytoplasm to non-cytoplasmic locations.
Instead the polypeptide will be expressed within the cytoplasm in
form of inclusion bodies or in soluble form. Thus upon expression
the polypeptide can be isolated and purified by well-known
procedures from the cell, in particular from cell extract. The
present invention also provides for the use of said vectors for the
regulated intracellular expression of a heterologous nucleic acid
sequence in a prokaryotic host cell; a prokaryotic host or
prokaryotic host cell transformed with said vector; a method for
the intracellular production of a heterologous polypeptide in a
prokaryotic host using said vector; and a vector for the
intracellular production of a heterologous polypeptide comprising a
promoter region, a heterologous nucleic acid sequence encoding a
heterologous polypeptide and a translation initiation region
consisting of the sequence AGGAGATATACAT.
[0109] In a preferred embodiment of the vector for the
intracellular expression the rhaBAD promoter consists of the
sequence depicted in SEQ ID No. 1, a sequence complementary thereof
and a variant sequence thereof. It is preferred that said rhaBAD
promoter region and said operably linked transcriptional unit
consist of the sequence depicted in SEQ ID No. 3 or SEQ ID No. 4, a
sequence complementary thereof or a variant sequence thereof.
According to the invention it is possible that the vector for
intracellular expression comprises a dicistronic transcriptional
unit. In another preferred embodiment of the invention the
transcriptional unit of the vector further comprises a translation
initiation region upstream of the initiation point of the
translation of said transcriptional unit, whereby the translation
initiation region consists of the sequence AGGAGATATACAT (SEQ ID
No. 2). In further preferred embodiments the vector for
intracellular expression comprises a transcription termination
region such as the rrnB transcriptional terminator sequence.
According to the invention the heterologous nucleic acid sequence
may encode a polypeptide such as an antibody, an antibody fragment
etc.
[0110] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications
without departing from the spirit or essential characteristics
thereof. The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations or any two or more of said steps or features. The
present disclosure is therefore to be considered as in all aspects
illustrated and not restrictive, the scope of the invention being
indicated by the appended Claims, and all changes which come within
the meaning and range of equivalency are intended to be embraced
therein. Various references are cited throughout this
Specification, each of which is incorporated herein by reference in
its entirety.
[0111] The foregoing description will be more fully understood with
reference to the following Examples. Such Examples, are, however,
exemplary of methods of practising the present invention and are
not intended to limit the scope of the invention.
EXAMPLES
Example 1
[0112] Construction of Expression Plasmids with Positively
Regulated Promoters
[0113] The Escherichia coli W3110 genome was scanned for positively
regulated operons. Based on the genomic data which are available on
the KEGG database (Kyoto Encyclopedia of Genes and Genomes,
http://www.genome.ad.jp/kegg/kegg2.html) positively regulated
catabolic promoters were identified and analysed for their use in
expression plasmids. The promoters should be tightly regulated and
induced by a cheap and non-toxic and therefore industrially useful
compound. The following promoters of different positively regulated
catabolic operons were chosen [0114] prp promoter (propionate
inducible) [0115] gutA promoter (glucitol inducible) [0116] melAB2
promoter (melibiose inducible)
[0117] The precise DNA fragments which contain the promoter
elements were selected based on the available information on the
corresponding regulator binding sites. Chromosomal DNA of
Escherichia coli was isolated by the method of Pitcher et al.,
1989, Letters in Applied Microbiology 8, 151-156. The promoter
fragments were amplified from the chromosomal DNA of strain W3110
by PCR using the following primers. The restriction sites of ClaI
and AflII are underlined. The sequences of the fragments are as
follows:
TABLE-US-00001 Pprp Pprp-5 5' aaa atc gat aaa tga aac gca tat ttg
3' Pprp-3 5' aaa ctt aag ttg tta tca act tgt tat 3'
AAAATCGATAACTGAAACGCATATTTGCGGATTAGTTCATGACTTTATCTCTAACAAA
TTGAAATTAAACATTTAATTTTATTAAGGCAATTGTGGCACACCCCTTGCTTTGTCTTT
ATCAACGCAAATAACAAGTTGATAACAACTTAAGTTT PgutA PgutA-5 5' aaa atc gat
gca tca cgc ccc gca caa 3' PgutA-3 5' aaa ctt aag tca gga ttt att
gtt tta 3' AAAATCGATGCATCACGCCCCGCACAAGGAAGCGGTAGTCACTGCCCGATACGGAC
TTTACATAACTCAACTCATTCCCCTCGCTATCCTTTTATTCAAACTTTCAAATTAAAATA
TTTATCTTTCATTTTGCGATCAAAATAACACTTTTAAATCTTTCAATCTGATTAGATTAG
GTTGCCGTTTGGTAATAAAACAATAAATCCTGACTTAAGTTT PmelAB2 PmelAB-5-1 5'
aaa atc gat gac tgc gag tgg gag cac 3' PmelAB-3 5' aaa ctt aag ggc
ttg ctt gaa taa ctt 3' MeIR CRP ##STR00001## ##STR00002##
##STR00003## GATTCGCCTGCCATGATGAAGTTATTCAAGCAAGCCCTTAAGTTT +1
[0118] (Binding site for CRP 2 is highlighted in light grey and
binding sites for MelR are highlighted in black)
[0119] The fragments were separated by agarose gelelectrophoresis
and isolated by the gelextraction kit QiaexII from Qiagen (Hilden,
Germany). The isolated fragments were cut with ClaI and AflII and
ligated to ClaI/AflII-cut pBW22 (Wilms et al., 2001, Biotechnology
and Bioengineering, 73 (2), 95-103). The resulting plasmids
containing the prp promoter (pBLL5), the gutA promoter (pBLL6) and
the melAB2 promoter (pBLL7) are identical except for the promoter
region ligated. The sequence of the inserted promoter fragments
were confirmed by sequencing (Microsynth GmbH, Balgach,
Switzerland).
Example 2
[0120] Construction of Fab Fragment Expression Plasmids
[0121] As an alternative to an IPTG-inducible lac promoter (plasmid
pMx9-HuCAL-Fab-H, Knappik et al., 1985, Gene 33, 103-119),
different positively regulated expression systems were analysed for
their capacity to produce Fab-H antibody fragments. The Fab-H
fragment was amplified out of plasmid pMx9-HuCAL-Fab-H by PCR using
the primers Fab-5 (5'-aaa cat atg aaa aag aca gct atc-3') and Fab-3
(5'-aaa aag ctt tta tca gct ttt cgg ttc-3'). The PCR-fragment was
cut with NdeI and HindIII and inserted into NdeI/HindIII-cut pBW22
(Volff et al., 1996, Mol. Microbiol. 21, 1037-1047) to create
plasmid pBW22-Fab-H (FIG. 1) containing the rhamnose inducible
rhaBAD promoter (SEQ ID NO. 1). The same PCR-fragment was inserted
into the different expression plasmids with inducible promoters.
The resulting Fab-H containing (putative) expression plasmids are
pBLL13 containing the prp promoter, pBLL14 containing the gutA
promoter and pBLL15 containing the melAB2 promoter (FIG. 2). The
sequence of the Fab-H insert of plasmid pBW22-Fab-H was confirmed
by sequencing.
Example 3
[0122] Expression of Fab Fragment
[0123] Strain W3110 (DSM 5911, Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was
transformed with the different expression plasmids. The plasmids
were isolated from clones which resulted from the different
transformations and checked via restriction analysis. Except
plasmid pBLL14 all plasmids had the expected restriction pattern.
The re-isolated plasmid pBLL14 showed an altered size and
restriction pattern which was suggested to be due to recombination
events. Therefore strain W3110 (pBLL14) was not tested in the
following assays. The remaining strains were tested for their
ability to secrete actively folded Fab-H antibody fragments. This
productivity test was performed as described in example 4. The
following inducers were added in a concentration of 0.2%
TABLE-US-00002 pBW22-Fab-H L(+)-Rhamnose monohydrate pBLL13 Sodium
propionate pBLL15 D(+)-Melibiose monohydrate D(+)-Raffinose
monohydrate D(+)-Galactose
[0124] The results from the dot blot experiments are shown in FIG.
3.
[0125] The rhamnose- and melibiose-induced strains W3110
(pBW22-Fab-H) and W3110 (pBLL15) showed promising dot blot results:
increasing signals over time and almost no background activity. The
portion of actively folded antibody fragments was quantified via
ELISA. The results are summarized in the following Table 1.
TABLE-US-00003 TABLE 1 ELISA results of the W3110 derivatives with
the different expression plasmids. The time after induction is
indicated. The uninduced cultures after 22 or 25 h were measured as
uninduced controls and the results from strain W3110
(pMx9-HuCAL-Fab-H) and TG1F'-(pMx9-HuCAL-Fab-H) are used as
references. The Fab-H concentration is given in mg/100 OD.sub.600/L
(n.d. not determined) 8 h 11.5/12 h 22/25 h 22/25 h Plasmid Inducer
induced uninduced in TG1F'- pMx9-HuCAL- IPTG nd Nd 68.64 84.56
Fab-H in W3110 pMx9-HuCAL- IPTG nd Nd 140.56 8.14 Fab-H pBW22-Fab-H
Rhamnose 176.88 259.56 328.62 6.52 pBLL13 Propionate nd 0.84 0.90
3.94 pBLL15 Melibiose 2.89 145.10 504.28 4.28
[0126] All strains grew well without any growth inhibition in the
presence or absence of the corresponding inducer up to OD.sub.600
between 4 and 6. The expression plasmids pBW22-Fab-H (containing
SEQ ID NO. 3) and pBLL15 led to the highest antibody fragment
titers after overnight induction. The melibiose induced strain
W3110 (pBLL15) showed a delayed increase in the formation of active
antibody fragments compared to the rhamnose (pBW22-Fab-H) induced
system.
[0127] The rhamnose inducible strain W3110 (pBW22-Fab-H) was tested
in the Respiration Activity Monitoring System (RAMOS, ACBiotec,
Julich, Germany), a novel measuring system for the on-line
determination of respiration activities in shake flasks. In
comparison to the normal shake flask experiment the antibody titer
(which was measured via ELISA) doubled (703.64 mg/L/100 OD.sub.600
after 23 h of induction). The optimised growth using the RAMOS
equipment favours the production of active antibody fragments.
Example 4
[0128] Melibiose Induction in Shake Flasks
[0129] E. coli W3110 carrying plasmid pBLL15 was tested for its
capacity to produce actively folded Fab-H antibody fragments.
Overnight cultures [in NYB medium (10 g/l tryptone, 5 g/l yeast
extract, 5 g/l sodium chloride) supplemented with 100 .mu.g/ml of
Ampicillin, 37.degree. C.] were diluted (1:50) in 20 ml of fresh
glycerol medium (as described by Kortz et al., 1995, J. Biotechnol.
39, 59-65, whereas the vitamin solution was used as described by
Kulla et al., 1983, Arch. Microbiol, 135, 1 and incubated at
30.degree. C. Melibose (0.2%) was added when the cultures reached
an OD.sub.600 of about 0.4. Samples (1 ml) were taken at different
time intervals, centrifuged and the pellets were stored at
-20.degree. C. The frozen cells were lysed according to the above
described lysozyme treatment and the supernatants were analysed in
dot blot and ELISA assays. 504,28 mg/L/100 OD.sub.600 of functional
Fab-H antibody fragments were obtained.
Example 5
[0130] Occurence of High Molecular Weight Aggregates
[0131] In order to find out if high molecular weight aggregates are
produced, western blot of extracts of strain W3110 (pBLL15), which
showed the highest antibody titer (Table 1), was conducted using
the anti-human Fab-H+AP conjugate. The culture was performed as
described in example 4. Samples were taken after 9, 12 and 23 hours
after induction with melibiose. Lower concentrations of high
molecular weight aggregates correspond to higher titers of
functional antibody fragments. The choice of the expression system
seems to influence the way in which the antibody fragments are
formed: functional or in aggregates.
Example 6
[0132] Influence of Signal Peptides
[0133] The genome database of E. coli was used to look for useful
signal peptides that could be used in combination with the Fab-H
fragments VL3-CL and VH-CH. The signal sequences from periplasmic
binding proteins for sugars, amino acids, vitamins and ions were
chosen. These periplasmic proteins represent a relatively
homogeneous group that have been more extensively studied than
other periplasmic proteins. Since they are generally abundant their
signal sequences have to ensure an efficient transport over the
inner membrane into the periplasm. All possible signal peptide Fab
combinations were checked for their sequence peptide and cleavage
site probability using the SignalP web server
(http://www.cbs.dtu.dk/services/SignalP-2.0/#submission) as shown
in the following Table 2.
TABLE-US-00004 Signal peptide Signal Max peptide Cleavage proba-
proba- bility bility OmpA (E. coli) - Outer membrane protein a
precursor MKKTA IAIAV ALAGF ATVAQ A APKDN (OmpA) 1.000 0.993 MKKTA
IAIAV ALAGF ATVAQ A DIELT (OmpA-VL3-CL, Fab-H) 1.000 0.971 PhoA (E.
coli) - Alkaline phosphatase precursor VKQST IALAL LPLLF TPVTK A
RTPEM (PhoA) 0.996 0.765 MKQST IALAL LPLLF TPVTK A QVQLK
(PhoA-VH-CH, Fab-H) 0.999 0.784 PelB (Erwinia chrysantemi) -
Pectate lyase precursor MKSLI TPITA GLLLA LSQPL LA ATDTG (PelB)
1.000 0.999 MKSLI TPITA GLLLA LSQPL LA DIELT (PelB-VL3-CL, Fab-H)
1.000 0.998 MKSLI TPITA GLLLA LSQPL LA QVQLK (PelB-VH-CH, Fab-H)
1.000 0.998 PelB (Erwinia carotovora) - Pectate lyase precursor
MKYLL PTAAA GLLLL AAQPA MA ANTGG (PelB) 1.000 1.000 MKYLL PTAAA
GLLLL AAQPA MA DIELT (PelB-VL3-CL, Fab-H) 1.000 1.000 MKYLL PTAAA
GLLLL AAQPA MA QVQLK (PelB-VH-CH, Fab-H) 1.000 1.000 PelB
(Xanthomonas campestris) - Pectate lyase precursor MKPKF STAAA
ASLFV GSLLV IGVAS A DPALE (PelB) 1.000 0.993 MKPKF STAAA ASLFV
GSLLV IGVAS A DIELT (PelB-VL3-CL, Fab-H) 1.000 0.985 MKPKF STAAA
ASLFV GSLLV IGVAS A QVQLK (PelB-VH-CH, Fab-H) 1.000 0.988 LamB (E.
coli) - Maltoporin precursor (Lambda receptor protein) MMITL RKLPL
AVAVA AGVMS AQAMA VDFHG (LamB) 1.000 0.975 MMITL RKLPL AVAVA AGVMS
AQAMA DIELT (LamB-VL3-CL, Fab-H) 1.000 0.979 MMITL RKLPL AVAVA
AGVMS AQAMA QVQLK (LamB-VH-CH, Fab-H) 1.000 0.988 MalE (E. coli) -
Maltose-binding protein precursor MKIKT GARIL ALSAL TTMMF SASAL A
KIEEG (MalE) 1.000 0.956 MKIKT GARIL ALSAL TTMMF SASAL A DIELT
(MalE-VL3-CL, Fab-H) 1.000 0.978 MKIKT GARIL ALSAL TTMMF SASAL A
QVQLK (MalE-VH-CH, Fab-H) 1.000 0.990 Bla (pBR322) (E. coli) -
Beta-lactamase MSIQH FRVAL IPFFA AFCLP VFA HPETL (Bla) 1.000 1.000
MSIQH FRVAL IPFFA AFCLP VFA DIELT (Bla-VL3-CL, Fab-H) 1.000 1.000
MSIQH FRVAL IPFFA AFCLP VFA QVQLK (Bla-VH-CH, Fab-H) 1.000 0.999
OppA (E. coli) - Periplasmic oligopeptide-binding protein MTNIT
KRSLV AAGVL AALMA GNVAL A ADVPA (OppA) 1.000 0.996 MTNIT KRSLV
AAGVL AALMA GNVAL A DIELT (OppA-VL3-CL, Fab-H) 1.000 0.911 MTNIT
KRSLV AAGVL AALMA GNVAL A QVQLK (OppA-VH-CH, Fab-H) 1.000 0.984
TreA (E. coli) - Periplasmic trehalase precursor (Alpha-trehalose
glucohydrolase MKSPA PSRPQ KMALI PACIF LCFAA LSVQA EETPV (TreA)
1.000 0.996 MKSPA PSRPQ KMALI PACIF LCFAA LSVQA DIELT (TreA-VL3-CL,
Fab-H) 1.000 0.961 MKSPA PSRPQ KMALI PACIF LCFAA LSVQA QVQLK
(TreA-VH-CH, Fab-H) 1.000 0.989 MppA (E. coli) - Periplasmic murein
peptide-binding protein precursor MKHSV SVTCC ALLVS SISLS YA AEVPS
(MppA) 1.000 0.943 MKHSV SVTCC ALLVS SISLS YA DIELT (MppA-VL3-CL,
Fab-H) 1.000 0.906 MKHSV SVTCC ALLVS SISLS YA QVQLK (MppA-VH-CH,
Fab-H) 1.000 0.938 BglX (E. coli) - Periplasmic beta-glucosidase
precursor MKWLC SVGIA VSLAL QPALA DDLFG (BglX) 1.000 0.999 MKWLC
SVGIA VSLAL QPALA DIELT (BglX-VL3-CL, Fab-H) 0.999 0.999 MKWLC
SVGIA VSLAL QPALA QVQLK (BglX-VH-CH, Fab-H) 1.000 0.996 ArgT (E.
coli) - Lysine-arginine-ornithine-binding periplasmic protein
precursor MKKSI LALSL LVGLS TAASS YA ALPET 1.000 0.929 MKKSI LALSL
LVGLS TAASS YA DIELT (ArgT-VL3-CL, Fab-H) 1.000 0.947 MKKSI LALSL
LVGLS TAASS YA QVQLK (ArgT-VH-CH, Fab-H) 1.000 0.960 MalS (E. coli)
- Alpha-amylase precursor MKLAA CFLTL LPGFA VA ASWTS (MalS) 1.000
0.794 MKLAA CFLTL LPGFA VA DIELT (MalS-VL3-CL, Fab-H) 0.998 0.995
MKLAA CFLTL LPGFA VA QVQLK (MalS-VH-CH, Fab-H) 1.000 0.990 HisJ (E.
coli) - Histidine-binding periplasmic protein precursor MKKLV LSLSL
VLAFS SATAA FA AIPQN (HisJ) 1.000 0.994 MKKLV LSLSL VLAFS SATAA FA
DIELT (HisJ-VL3-CL, Fab-H) 1.000 0.957 MKKLV LSLSL VLAFS SATAA FA
QVQLK (HisJ-VH-CH, Fab-H) 1.000 0.988 XylF (E. coli) -
D-Xylose-binding periplasmic protein precursor MKIKN ILLTL CTSLL
LTNVA AHA KEVKI (XylF) 1.000 0.996 MKIKN ILLTL CTSLL LTNVA AHA
DIELT (XylF-VL3-CL, FabH) 1.000 0.992 MKIKN ILLTL CTSLL LTNVA AHA
QVQLK (XylF-VH-CH, Fab-H) 1.000 0.996 FecB (E. coli) - Iron(III)
dicitrate-binding periplasmic protein precursor MLAFI RFLFA GLLLV
ISHAF A ATVQD (FecB) 1.000 0.975 MLAFI RFLFA GLLLV ISHAF A DIELT
(FecB-VL3-CL, Fab-H) 1.000 0.989 MLAFI RFLFA GLLLV ISHAF A QVQLK
(FecB-VH-CH, Fab-H) 1.000 0.990
[0134] The following six combinations were chosen: [0135]
LamB-VL3-CL (Maltoporin precursor) [0136] MalE-VH-CH
(Maltose-binding protein precursor) [0137] Bla-VL3-CL
(Beta-lactamase) [0138] TreA-VH-CH (Periplasmic trehalase
precursor) [0139] ArgT-VL3-CL (Lysine-arginine-ornithine-binding
periplasmic protein precursor) [0140] FecB-VH-CH (Iron (III)
dicitrate-binding periplasmic protein precursor
[0141] The gene fusions to generate signal peptide (SP) to VL3-CL
and VH-CH fusions were carried out with overlapping PCR primers and
are summarized in the following amplification Table 3
TABLE-US-00005 Primer Template Fragment LamB-VL3-CL lamB-5 Genomic
DNA of E. coli lamB-SP lamB-3 W3110 lamB-VL3-5 pMx9-HuCAL-Fab-H-S-S
VL3-CL VL3-3 lamB-5 lamB-SP/VL3-CL lamB-VL3-CL VL3-3 MalE-VH-CH
malE-5 Genomic DNA of E. coli malE-SP malE-3 W3110 malE-VH-CH
pMx9-HuCAL-Fab-H VH-CH VH-3 malE-5 malE-SP/VH-CH malE-VH-CH VL3-3
Bla-VL3-CL bla-5 Genomic DNA of E. coli bla-SP bla-3 W3110
bla-VL3-5 pMx9-HuCAL-Fab-H-S-S VL3-CL VL3-3 bla-5 bla-SP/VL3-CL
bla-VL3-CL VL3-3 TreA-VH-CH treA-5 Genomic DNA of E. coli treA-SP
treA-3 W3110 treA-VH-CH pMx9-HuCAL-Fab-H-S-S VH-CH VH-3 treA-5
treA-SP/VH-CH treA-VH-CH VH-3 ArgT-VL3-CL argT-5 Genomic DNA of E.
coli argT-SP argT-3 W3110 argT-VL3-5 pMx9-HuCAL-Fab-H VL3-CL VL3-3
argT-5 argT-SP/VL3-CL argT-VL3-CL VL3-3 FecB-VH-CH fecB-5 Genomic
DNA of E. coli fecB-SP fecB-3 W3110 fecB-VH-CH pMx9-HuCAL-Fab-H-S-S
VH-CH VL3-3 fecB-5 fecB-SP/VH-CH fecB-VH-CH VL3-3
[0142] The fusions of the signal peptide sequences with the VL3-CL
and VH-CH sequences were performed as described elsewhere (Horton,
R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R.
(1989) Engineering hybrid genes without the use of restriction
enzymes: gene splicing by overlap extension. Gene 77, 61-68). The
SP-VL3-CL genes were cut with restriction enzymes NdeI and PstI and
ligated into NdeI/PstI cut pBW22 and into pBLL7. The resulting
plasmids were cut with PstI and HindIII and ligated to PstI/HindIII
cut SP-VH-CH genes. Since the integration of the bla-VL3-CL and
fecB-VH-CH genes was not possible only the Fab-H expression plasmid
containing the lamB-VL3-CL and malE-VH-CH genes could be tested. A
lamB-VL3-CL/malE-VH-CH expression plasmid containing the rhamnose
inducible promoter (pAKL14) was obtained. The
lamB-VL3-CL/malE-VH-CH genes which were isolated from plasmid
pAKL15 (example 7) as AflII/HindIII fragment were ligated into
AflII/HindIII-cut pBLL7 to obtain pAKL15E. FIGS. 4 and 8 illustrate
the lamB-VL3-CL/malE-VH-CH expression plasmids pAKL14 and
pAKL15E.
Example 7
[0143] Influence of Translation Initiation Regions on Fab
Expression
[0144] The Fab-H genes of plasmid pAKL14 (containing SEQ ID NO. 4)
and plasmid pAKL15E contain the same DNA sequence 5' of the start
codon (translation initiation region) whereas in the original
plasmid pMx9-HuCAL-Fab-H the translation initiation regions of both
Fab-H genes are different. A comparison of the translation
initiation regions sequences of plasmid pMx9-HuCAL-Fab-H and
pAKL14/pAKL15E is shown in the following Table 4:
TABLE-US-00006 pMx9-HuCAL-Fab-H ompA-VL3-CL gagggcaaaaa atg
phoA-VH-CH aggagaaaataaa atg pAKL14/pAKL15E lamB-VL3-CL
aggagatatacat atg malE-VH-CH aggagatatacat atg
[0145] The productivity of strain W3110 (pAKL14) was tested in
shake flasks as described in example 4. The strain grew well in the
presence or absence of L-rhamnose. That means the production of
Fab-H did not influence the viability of the cells. As shown in
FIG. 5 the dot blot results looked promising.
[0146] To analyse the presence of non-reducible high molecular
weight aggregates a Western blot was performed (FIG. 6). Although
high molecular weight aggregates appear after an induction time of
5 hours their amount only slightly increases after 23 h. The
non-induced culture shows high molecular weight bands which might
be due to a weak unspecific background production. The
corresponding ELISA values are given in the following Table 5
(Fab-H concentration (mg/L/100 OD.sub.600) in lysozyme extracts of
uninduced and rhamnose induced strain W3110 (pAKL14)).
TABLE-US-00007 5 h 7 h 12 h 23 h 23 h Plasmid Inducer induced
uninduced in W3110 Rhamnose 29.04 267.48 308.40 596.14 2.84
[0147] The new signal peptide constructs (in combination with the
modified translation initiation signals) again increased the
antibody fragment titer from 328.62 mg/L/100 OD.sub.600 (plasmid
pBW22-Fab-H which contains the MOR gene construct from plasmid
pMx9-HuCAL-Fab-H) to 596.14 mg/L/100 OD.sub.600 plasmid pAKL14) and
to 878.86 mg/L/100 OD.sub.600 (plasmid pAKL15E). The sequencing of
the lamB-VL3-CL and malE-VH-CH genes in pAKL14 revealed three base
exchanges which are supposed to be due to the construction of the
fusion genes by two consecutive PCR reactions. The base exchanges
led to the following amino acid changes (the wrong amino acids are
emphasized):
TABLE-US-00008 VL3-CL (pAKL14) - pI = 4.85
MMITLRKLPLAVAVAAGVMSAQAMADIELTQPPSVSVAPGQTARISCSGN
ALGDKYASWYQQNPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTIS
GTQAEDEADYYCQSYDSPQVVFGGGTKLTVLGQPKAAPSVTLFPPSSEEL
QANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS
SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEA VH-CH (pAKL14) - p1 = 9.52
MKIKTGARILALSALTTMMFSASALAQVQLKESGPALVKPTQTLTLTCTF
SGFSLSTSGVGVGWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKD
TSKNQVVLTMTNMDPVDTATYYCARYPVTQRSYMDVWGQGTLVTVSSAST
KGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
[0148] The light chain of Fab-H carries two mistakes (D50N, K63N)
and the heavy chain one amino acid exchange (F156L). To restore the
original Fab-H sequence two fragments from plasmid pAKL14 (138 bp
SexAI/BamHI and 310 bp BssHII/HindIII fragment) were exchanged
against the homologous fragments of plasmid pBW22-Fab-H (which
carries the unchanged Fab-H gene sequence). The resulting plasmid
pAKL15 carries the correct Fab-H sequence. The exchange of the
three amino acids had no apparent effect on the overall Fab-H
properties since the pI was unchanged. Therefore the capacity of
strain W3110 (pAKL15) to produce functional Fab-H antibody
fragments was supposed to be similar to strain W3110 (pAKL14) and
was not analysed.
[0149] The Fab-H antibody fragment productivity could be increased
by using different optimisation strategies. The following Table 6
summarizes the improvements:
TABLE-US-00009 Concentration of functional Fab-H Antibody (mg/L/
Activity Strain Improvement 100 OD.sub.600) increase TG1F'- MOR
strain 84.56 (pMx9-HuCAL- Fab-H) W3110 Strain background 140.45 1.7
(pMx9-HuCAL- Fab-H) W3110 Expression system 328.62 3.9
(pBW22-Fab-H) (Rhamnose) W3110 (pBLL15) Expression system 504.28 6
(Melibiose) W3110 (pAKL14) Signal peptide 596.14 7 Translation
(Rhamnose) W3110 (pAKL15E) Signal peptide 878.86 10.4 Translation
(Melibiose)
[0150] Strains which produced high Fab-H antibody titers were
analysed via SDS-PAGE (FIG. 7). The highest functional Fab-H
concentrations were measured in strains which produce a balanced
amount of light and heavy chain (lanes 4 and 5). The rhamnose
inducible strains which carry the Fab-H fragment such as W3110
(pBW22-Fab-H) (lane 3) strongly overproduce the light chain.
Example 8
[0151] Melibiose Induction in Shake Flasks
[0152] E. coli W3110 carrying plasmid pAKL15E (FIG. 8) was tested
for its capacity to produce actively folded Fab-H antibody
fragments. Overnight cultures [in NYB medium (10 g/l tryptone, 5
g/l yeast extract, 5 g/l sodium chloride) supplemented with 100
.mu.g/ml of Ampicillin, 37.degree. C.] were diluted (1:50) in 20 ml
of fresh glycerol medium (as described by Kortz et al., 1995, J.
Biotechnol. 39, 59-65, whereas the vitamin solution was used as
described by Kulla et al., 1983, Arch. Microbiol, 135, 1 and
incubated at 30.degree. C. Melibose (0.2%) was added when the
cultures reached an OD.sub.600 of about 0.4. Samples (1 ml) were
taken at different time intervals, centrifuged and the pellets were
stored at -20.degree. C. The frozen cells were lysed according to
the above described lysozyme treatment and the supernatants were
analysed in SDS-PAGE and ELISA assays. The melibiose inducible
strain which carry the Fab-H genes with the altered signal peptides
(lamB-VL3-CL/malE-VH-CH) showed the highest Fab-H antibody titers
(Table 6). The light and heavy chain of Fab-H were produced in
equal amounts (FIG. 9).
Example 9
[0153] Intracellular Production of Antibody Fragments
[0154] Origami host strains provide mutations in both the
thioredoxin reductase (trxB) and glutathione reductase (gor) genes,
enhancing disulfide bond formation and permit protein folding in
the bacterial cytoplasm. To construct the VL3-CL and VH-CH genes
without signal peptide regions the following primers were used:
TABLE-US-00010 5'-VL 5'-aaa cat atg gat atc gaa ctg acc cag-3'
(NdeI restriction site) 3'-CL 5'-aaa ctg cag tta tca ggc ctc agt
cgg-3' (PstI restriction site) 5'-VH 5'-aaa ctg cag gag ata tac ata
tgc agg tgc aat tga a-3' (PstI restriction site) 3'-CH 5'-aaa aag
ctt tta tea gct ttt cgg ttc-3' (HindIII restriction site)
[0155] The corresponding VL3-CL and VH-CH genes were amplified and
checked via restriction analysis. The NdeI/PstI cut VL3-CH fragment
was integrated into NdeI/PstI cut plasmid pBW22. The resulting
plasmid was cut with PstI and HindIII and ligated to the
PstI/HindIII cut VH-CH fragment to get plasmid pJKL6. The plasmid
was transformed into the Origami strain and strain W3110 as a
reference. The productivity of the strains W3110 (pJKL6) and
Origami (pJKL6) was tested in shake flasks as described in example
4.
[0156] To analyse the presence of functional antibody fragments and
non-reducible high molecular weight aggregates a Western blot was
performed. Both strains hardly produce any functional antibody
fragments. Strain W3110 accumulates high molecular weight
aggregates with increasing induction times (W3110) whereas the
Origami strain does not produce any antibody fragments. The
corresponding ELISA values are given in the following Table 7
(Fab-H concentration (mg/L/100 OD.sub.600) in lysozyme extracts of
uninduced and rhamnose induced strains Origami (pJKL6) and W3110
(pJKL6)):
TABLE-US-00011 7 h 11 h 24 h 24 h Plasmid Inducer induced uninduced
Origami pJKL6 Rhamnose 5.24 6.86 10.54 2.60 W3110 pJKL6 Rhamnose
2.73 5.34 5.2 2.83
Example 10
[0157] Rhamnose Induction of a Single Chain Antibody (scFv, S1) in
Shake Flasks
[0158] The scFv gene was isolated via PCR using the primers 5-S
(5'-aaa cat atg aaa tac cta ttg cct acg gc-3') and 3-S1 (5'-aaa aag
ctt act acg agg aga cgg-3'). The corresponding S1 protein contains
a PelB signal sequence which is responsible for transport of the
protein to the periplasm of E. coli. The PCR-fragment was cut with
NdeI and HindIII and inserted into NdeI/HindIII-cut pBW22 to create
plasmid pBW22-pelB-S1 containing the rhamnose inducible rhaBAD
promoter (FIG. 10). The sequence of the S1 insert of plasmid
pBW22-pelB-S1 was confirmed by sequencing. Strain W3110 (DSM 5911,
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany) was transformed with plasmid pBW22-pelB-S1.
The plasmids were isolated from different clones and verified by
restriction analysis. E. coli W3110 (pBW22-pelB-S1) was tested for
its capacity to produce soluble S1. Overnight cultures [in NYB
medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l sodium
chloride) supplemented with 100 .mu.g/ml of Ampicillin, 37.degree.
C.] were diluted (1:50) in 20 ml of fresh glycerol medium [as
described by Kortz et al., 1995, J. Biotechnol. 39, 59-65, with the
exception of the vitamin solution (as described by Kulla et al.,
1983, Arch. Microbiol, 135, 1)] and incubated at 30.degree. C.
Rhamnose (0.2%) was added when the cultures reached an OD.sub.600
of about 0.4. Samples (1 ml) were taken at different time
intervals, centrifuged and the pellets were stored at -20.degree.
C. The frozen cells were lysed according to the above described
lysozyme treatment and the supernatants and insoluble protein
pellets were analysed via SDS-PAGE (FIG. 11) and Bioanalyzer. Most
of the S1 protein (mg/L/100 OD.sub.600) was produced in the soluble
protein fraction.
Example 11
[0159] Construction of a Broad-Host-Range Rhamnose Expression
Plasmid for Pseudomonas and Related Bacteria
[0160] The following cloning experiments were performed in
Escherichia coli JM109. The broad-host-range cloning vector
pBBR1MCS-2 (NCBI accession number U23751) was cut with AgeI/NsiI.
The lacZ.alpha. gene was deleted and replaced by the
oligonucleotides 3802 (5'-tgt taa ctg cag gat cca agc tta-3') and
3803 (5'-ccg gta agc ttg gat cct gca gtt aac atg ca-3') to get
plasmid pJOE4776.1. The rhaRSP fragment was provided by plasmid
pJKS408 (unpublished) which contains the genomic rhaRS fragment (2
kb) of Escherichia coli JM109. Plasmid pJKS408 was cut with
BamHI/HindIII and ligated to the BamHI/HindIII cut eGFP fragment
(0.7 kb) of plasmid pTST101 [Stumpp, T., Wilms, B., Altenbuchner,
J. (2000): Ein neues, L-Rhamnose-induzierbares Expressionssystem
fur Escherichia coli. Biospectrum 6, 33-36]. The rhaRSPmalE-eGFP
fragment (4 kb) was isolated via NsiI/HindIII from the resulting
plasmid pJOE4030.2 and integrated into NsiI/HindIII cut pJOE4776.1.
Plasmid pJOE4776.1 (FIG. 12) contains the rhaBAD promoter region in
combination with the genes of the regulatory proteins RhaS and RhaR
of the rhamnose operon of Escherichia coli in a broad-host-range
plasmid backbone.
Example 12
[0161] Rhamnose Induction of a Nitrilase in Shake Flasks
[0162] The nitrilase gene was cut with NdeI and BamHI from Plasmid
pDC12 (Kiziak et al., 2005) and inserted into NdeI/BamHI-cut
pJOE4782.1 to create plasmid pAKLP2 containing the L-rhamnose
inducible rhaBAD promoter (FIG. 13). E. coli XL1-Blue was
transformed with plasmid pAKLP2 as an intermediate step. The
plasmids were isolated from different clones and verified by
restriction analysis. Pseudomonas putida KT-2440 (DSM 6125,
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany) was transformed with the isolated plasmid
pAKLP2 from E. coli XL1blue(pAKLP2). Pseudomonas putida KT-2440
(pAKLP2) was tested for its capacity to produce nitrilase.
Overnight cultures [in NYB medium (10 g/l tryptone, 5 g/l yeast
extract, 5 g/l sodium chloride) supplemented with 50 .mu.g/ml of
Kanamycin, 30.degree. C.] were diluted in 20 ml of fresh glycerol
medium [as described by Kortz et al., 1995, J. Biotechnol. 39,
59-65, with the exception of the vitamin solution (as described by
Kulla et al., 1983, Arch. Microbiol, 135, 1)] to an OD.sub.600 of
about 0.1 and incubated at 30.degree. C. L-rhamnose (1.0%) was
added when the cultures reached an OD.sub.600 of about 0.25.
Samples (1 ml) were taken at different time intervals, centrifuged
and the pellets were stored at -20.degree. C. The pellets were
resuspended in Tris/HCl-buffer (50 mM, pH 8.0) and the cell
suspensions were analysed via SDS-PAGE (FIG. 14).
Example 13
[0163] L-rhamnose Induction of a Fragment Antibody (FabM) in Shake
Flasks
[0164] The Fab-M gene was cut with NdeI and BamHI from plasmid
pBW22-FabM and inserted into NdeI/BamHI-cut pJOE4782.1 to create
plasmid pAKLP1 containing the L-rhamnose inducible rhaBAD promoter
(FIG. 15). E. coli XL1-Blue was transformed with plasmid pAKLP1 as
an intermediate step. The plasmids were isolated from different
clones and verified by restriction analysis. Pseudomonas putida
KT-2440 (DSM 6125, Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH, Braunschweig, Germany) was transformed with the
isolated plasmid pAKLP1 from E. coli XL1blue(pAKLP1). Pseudomonas
putida KT-2440 (pAKLP1) was tested for its capacity to produce
Fab-M. Overnight cultures [in NYB medium (10 g/l tryptone, 5 g/l
yeast extract, 5 g/l sodium chloride) supplemented with 50 .mu.g/ml
of Kanamycin, 30.degree. C.] were diluted in 20 ml of fresh
glycerol medium [as described by Kortz et al., 1995, J. Biotechnol.
39, 59-65, with the exception of the vitamin solution (as described
by Kulla et al., 1983, Arch. Microbiol, 135, 1)] to an OD.sub.600
of about 0.1 and incubated at 30.degree. C. L-rhamnose (1.0%) was
added when the cultures reached an OD.sub.600 of about 0.25.
Samples (1 ml) were taken at different time intervals, centrifuged
and the pellets were stored at -20.degree. C. The pellets were
resuspended in Tris/HCl-buffer (50 mM, pH 8.0) and the cell
suspensions were analysed via SDS-PAGE (FIG. 16).
Example 14
[0165] Single Chain Antibody Expression Using an Escherichia coli
Secretion System in High Cell Density Fermentation
[0166] Escherichia coli W3110 was transformed with plasmid
pBW22-pelB-S1. The plasmids were isolated from different clones and
verified by restriction analysis and one clone was used for further
experiments. Pre-cultures in shake flask were inoculated from
single colonies in Lonza's batch phase medium. The pre-culture was
used to inoculate a 20 L fermenter. Cells were grown according to
Lonza's high cell density fermentation regime. Samples (10 ml) of
the culture were taken at different time points before and after
rhamnose induction. Cells were separated from fermentation medium
by centrifugation at 10,000 g. SDS gel analysis of samples from the
cell free fermentation medium show a protein band at 28.4 kD with
increasing density. This protein is the single chain antibody S1
released from the growing culture into the fermentation medium. A
quantification of the S1 protein content with an Agilent 2100
Bioanalyser (Agilent, Palo Alto, USA) indicated an accumulation of
up to 2 g/L/100 OD.sub.600 S1 protein in the fermentation broth
after rhamnose induction. After lysozyme treatment of the cell
pellet, the insoluble protein pellet contained only traces of the
S1 protein whereas the soluble protein fraction (supernatant)
showed a strong S1 protein band, corresponding to about 1 g/L/100
OD.sub.600 (see FIG. 17).
Sequence CWU 1
1
751129DNAEscherichia coli 1caccacaatt cagcaaattg tgaacatcat
cacgttcatc tttccctggt tgccaatggc 60ccattttcct gtcagtaacg agaaggtcgc
gaattcaggc gctttttaga ctggtcgtaa 120tgaacaatt 129213DNAEscherichia
coli 2aggagatata cat 1331616DNAEscherichia coli 3atcgatcacc
acaattcagc aaattgtgaa catcatcacg ttcatctttc cctggttgcc 60aatggcccat
tttcctgtca gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg
120tcgtaatgaa caattcttaa gaaggagata tacatatgaa aaagacagct
atcgcgattg 180cagtggcact ggctggtttc gctaccgtag cgcaggccga
tatcgaactg acccagccgc 240cttcagtgag cgttgcacca ggtcagaccg
cgcgtatctc gtgtagcggc gatgcgctgg 300gcgataaata cgcgagctgg
taccagcaga aacccgggca ggcgccagtt ctggtgattt 360atgatgattc
tgaccgtccc tcaggcatcc cggaacgctt tagcggatcc aacagcggca
420acaccgcgac cctgaccatt agcggcactc aggcggaaga cgaagcggat
tattattgcc 480agagctatga ctctcctcag gttgtgtttg gcggcggcac
gaagttaacc gttcttggcc 540agccgaaagc cgcaccgagt gtgacgctgt
ttccgccgag cagcgaagaa ttgcaggcga 600acaaagcgac cctggtgtgc
ctgattagcg acttttatcc gggagccgtg acagtggcct 660ggaaggcaga
tagcagcccc gtcaaggcgg gagtggagac caccacaccc tccaaacaaa
720gcaacaacaa gtacgcggcc agcagctatc tgagcctgac gcctgagcag
tggaagtccc 780acagaagcta cagctgccag gtcacgcatg aggggagcac
cgtggaaaaa accgttgcgc 840cgactgaggc ctgataagca tgcgtaggag
aaaataaaat gaaacaaagc actattgcac 900tggcactctt accgttgctc
ttcacccctg ttaccaaagc ccaggtgcaa ttgaaagaaa 960gcggcccggc
cctggtgaaa ccgacccaaa ccctgaccct gacctgtacc ttttccggat
1020ttagcctgtc cacgtctggc gttggcgtgg gctggattcg ccagccgcct
gggaaagccc 1080tcgagtggct ggctctgatt gattgggatg atgataagta
ttatagcacc agcctgaaaa 1140cgcgtctgac cattagcaaa gatacttcga
aaaatcaggt ggtgctgact atgaccaaca 1200tggacccggt ggatacggcc
acctattatt gcgcgcgtta tcctgttact cagcgttctt 1260atatggatgt
ttggggccaa ggcaccctgg tgacggttag ctcagcgtcg accaaaggtc
1320caagcgtgtt tccgctggct ccgagcagca aaagcaccag cggcggcacg
gctgccctgg 1380gctgcctggt taaagattat ttcccggaac cagtcaccgt
gagctggaac agcggggcgc 1440tgaccagcgg cgtgcatacc tttccggcgg
tgctgcaaag cagcggcctg tatagcctga 1500gcagcgttgt gaccgtgccg
agcagcagct taggcactca gacctatatt tgcaacgtga 1560accataaacc
gagcaacacc aaagtggata aaaaagtgga accgaaaagc tgataa
161641639DNAEscherichia coli 4atcgatcacc acaattcagc aaattgtgaa
catcatcacg ttcatctttc cctggttgcc 60aatggcccat tttcctgtca gtaacgagaa
ggtcgcgaat tcaggcgctt tttagactgg 120tcgtaatgaa caattcttaa
gaaggagata tacatatgat gattactctg cgcaaacttc 180ctctggcggt
tgccgtcgca gcgggcgtaa tgtctgctca ggcaatggct gatatcgaac
240tgacccagcc gccttcagtg agcgttgcac caggtcagac cgcgcgtatc
tcgtgtagcg 300gcaatgcgct gggcgataaa tacgcgagct ggtaccagca
gaatcccggg caggcgccag 360ttctggtgat ttatgatgat tctgaccgtc
cctcaggcat cccggaacgc tttagcggat 420ccaacagcgg caacaccgcg
accctgacca ttagcggcac tcaggcggaa gacgaagcgg 480attattattg
ccagagctat gactctcctc aggttgtgtt tggcggcggc acgaagttaa
540ccgttcttgg ccagccgaaa gccgcaccga gtgtgacgct gtttccgccg
agcagcgaag 600aattgcaggc gaacaaagcg accctggtgt gcctgattag
cgacttttat ccgggagccg 660tgacagtggc ctggaaggca gatagcagcc
ccgtcaaggc gggagtggag accaccacac 720cctccaaaca aagcaacaac
aagtacgcgg ccagcagcta tctgagcctg acgcctgagc 780agtggaagtc
ccacagaagc tacagctgcc aggtcacgca tgaggggagc accgtggaaa
840aaaccgttgc gccgactgag gcctgataac tgcaggagat atacatatga
aaataaaaac 900aggtgcacgc atcctcgcat tatccgcatt aacgacgatg
atgttttccg cctcggctct 960cgcccaggtg caattgaaag aaagcggccc
ggccctggtg aaaccgaccc aaaccctgac 1020cctgacctgt accttttccg
gatttagcct gtccacgtct ggcgttggcg tgggctggat 1080tcgccagccg
cctgggaaag ccctcgagtg gctggctctg attgattggg atgatgataa
1140gtattatagc accagcctga aaacgcgtct gaccattagc aaagatactt
cgaaaaatca 1200ggtggtgctg actatgacca acatggaccc ggtggatacg
gccacctatt attgcgcgcg 1260ttatcctgtt actcagcgtt cttatatgga
tgtttggggc caaggcaccc tggtgacggt 1320tagctcagcg tcgaccaaag
gtccaagcgt gcttccgctg gctccgagca gcaaaagcac 1380cagcggcggc
acggctgccc tgggctgcct ggttaaagat tatttcccgg aaccagtcac
1440cgtgagctgg aacagcgggg cgctgaccag cggcgtgcat acctttccgg
cggtgctgca 1500aagcagcggc ctgtatagcc tgagcagcgt tgtgaccgtg
ccgagcagca gcttaggcac 1560tcagacctat atttgcaacg tgaaccataa
accgagcaac accaaagtgg ataaaaaagt 1620ggaaccgaaa agctgataa
1639527DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5aaaatcgata aatgaaacgc atatttg 27627DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6aaacttaagt tgttatcaac ttgttat 277154DNAEscherichia coli
7aaaatcgata actgaaacgc atatttgcgg attagttcat gactttatct ctaacaaatt
60gaaattaaac atttaatttt attaaggcaa ttgtggcaca ccccttgctt tgtctttatc
120aacgcaaata acaagttgat aacaacttaa gttt 154827DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8aaaatcgatg catcacgccc cgcacaa 27927DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9aaacttaagt caggatttat tgtttta 2710218DNAEscherichia coli
10aaaatcgatg catcacgccc cgcacaagga agcggtagtc actgcccgat acggacttta
60cataactcaa ctcattcccc tcgctatcct tttattcaaa ctttcaaatt aaaatattta
120tctttcattt tgcgatcaaa ataacacttt taaatctttc aatctgatta
gattaggttg 180ccgtttggta ataaaacaat aaatcctgac ttaagttt
2181127DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11aaaatcgatg actgcgagtg ggagcac
271227DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12aaacttaagg gcttgcttga ataactt
2713161DNAEscherichia coli 13aaaatcgata ctctgctttt caggtaattt
attcccataa actcagattt actgctgctt 60cacgcaggat ctgagtttat gggaatgctc
aacctggaag ccggaggttt tctgcagatt 120cgcctgccat gatgaagtta
ttcaagcaag cccttaagtt t 1611424DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 14aaacatatga aaaagacagc tatc
241527DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15aaaaagcttt tatcagcttt tcggttc
271626PRTEscherichia coli 16Met Lys Lys Thr Ala Ile Ala Ile Ala Val
Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Ala Pro Lys Asp
Asn20 251726PRTEscherichia coli 17Met Lys Lys Thr Ala Ile Ala Ile
Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Asp Ile
Glu Leu Thr20 251826PRTEscherichia coli 18Val Lys Gln Ser Thr Ile
Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr1 5 10 15Pro Val Thr Lys Ala
Arg Thr Pro Glu Met20 251926PRTEscherichia coli 19Met Lys Gln Ser
Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr1 5 10 15Pro Val Thr
Lys Ala Gln Val Gln Leu Lys20 252027PRTErwinia chrysanthemi 20Met
Lys Ser Leu Ile Thr Pro Ile Thr Ala Gly Leu Leu Leu Ala Leu1 5 10
15Ser Gln Pro Leu Leu Ala Ala Thr Asp Thr Gly20 252127PRTErwinia
chrysanthemi 21Met Lys Ser Leu Ile Thr Pro Ile Thr Ala Gly Leu Leu
Leu Ala Leu1 5 10 15Ser Gln Pro Leu Leu Ala Asp Ile Glu Leu Thr20
252227PRTErwinia chrysanthemi 22Met Lys Ser Leu Ile Thr Pro Ile Thr
Ala Gly Leu Leu Leu Ala Leu1 5 10 15Ser Gln Pro Leu Leu Ala Gln Val
Gln Leu Lys20 252327PRTErwinia carotovora 23Met Lys Tyr Leu Leu Pro
Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met
Ala Ala Asn Thr Gly Gly20 252427PRTErwinia carotovora 24Met Lys Tyr
Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln
Pro Ala Met Ala Asp Ile Glu Leu Thr20 252527PRTErwinia carotovora
25Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1
5 10 15Ala Gln Pro Ala Met Ala Gln Val Gln Leu Lys20
252631PRTXanthomonas campestris 26Met Lys Pro Lys Phe Ser Thr Ala
Ala Ala Ala Ser Leu Phe Val Gly1 5 10 15Ser Leu Leu Val Ile Gly Val
Ala Ser Ala Asp Pro Ala Leu Glu20 25 302731PRTXanthomonas
campestris 27Met Lys Pro Lys Phe Ser Thr Ala Ala Ala Ala Ser Leu
Phe Val Gly1 5 10 15Ser Leu Leu Val Ile Gly Val Ala Ser Ala Asp Ile
Glu Leu Thr20 25 302831PRTXanthomonas campestris 28Met Lys Pro Lys
Phe Ser Thr Ala Ala Ala Ala Ser Leu Phe Val Gly1 5 10 15Ser Leu Leu
Val Ile Gly Val Ala Ser Ala Gln Val Gln Leu Lys20 25
302930PRTEscherichia coli 29Met Met Ile Thr Leu Arg Lys Leu Pro Leu
Ala Val Ala Val Ala Ala1 5 10 15Gly Val Met Ser Ala Gln Ala Met Ala
Val Asp Phe His Gly20 25 303030PRTEscherichia coli 30Met Met Ile
Thr Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala1 5 10 15Gly Val
Met Ser Ala Gln Ala Met Ala Asp Ile Glu Leu Thr20 25
303130PRTEscherichia coli 31Met Met Ile Thr Leu Arg Lys Leu Pro Leu
Ala Val Ala Val Ala Ala1 5 10 15Gly Val Met Ser Ala Gln Ala Met Ala
Gln Val Gln Leu Lys20 25 303231PRTEscherichia coli 32Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr1 5 10 15Thr Met
Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly20 25
303331PRTEscherichia coli 33Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr1 5 10 15Thr Met Met Phe Ser Ala Ser Ala Leu
Ala Asp Ile Glu Leu Thr20 25 303431PRTEscherichia coli 34Met Lys
Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr1 5 10 15Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Gln Val Gln Leu Lys20 25
303528PRTEscherichia coli 35Met Ser Ile Gln His Phe Arg Val Ala Leu
Ile Pro Phe Phe Ala Ala1 5 10 15Phe Cys Leu Pro Val Phe Ala His Pro
Glu Thr Leu20 253628PRTEscherichia coli 36Met Ser Ile Gln His Phe
Arg Val Ala Leu Ile Pro Phe Phe Ala Ala1 5 10 15Phe Cys Leu Pro Val
Phe Ala Asp Ile Glu Leu Thr20 253728PRTEscherichia coli 37Met Ser
Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala1 5 10 15Phe
Cys Leu Pro Val Phe Ala Gln Val Gln Leu Lys20 253831PRTEscherichia
coli 38Met Thr Asn Ile Thr Lys Arg Ser Leu Val Ala Ala Gly Val Leu
Ala1 5 10 15Ala Leu Met Ala Gly Asn Val Ala Leu Ala Ala Asp Val Pro
Ala20 25 303931PRTEscherichia coli 39Met Thr Asn Ile Thr Lys Arg
Ser Leu Val Ala Ala Gly Val Leu Ala1 5 10 15Ala Leu Met Ala Gly Asn
Val Ala Leu Ala Asp Ile Glu Leu Thr20 25 304031PRTEscherichia coli
40Met Thr Asn Ile Thr Lys Arg Ser Leu Val Ala Ala Gly Val Leu Ala1
5 10 15Ala Leu Met Ala Gly Asn Val Ala Leu Ala Gln Val Gln Leu
Lys20 25 304135PRTEscherichia coli 41Met Lys Ser Pro Ala Pro Ser
Arg Pro Gln Lys Met Ala Leu Ile Pro1 5 10 15Ala Cys Ile Phe Leu Cys
Phe Ala Ala Leu Ser Val Gln Ala Glu Glu20 25 30Thr Pro
Val354235PRTEscherichia coli 42Met Lys Ser Pro Ala Pro Ser Arg Pro
Gln Lys Met Ala Leu Ile Pro1 5 10 15Ala Cys Ile Phe Leu Cys Phe Ala
Ala Leu Ser Val Gln Ala Asp Ile20 25 30Glu Leu
Thr354335PRTEscherichia coli 43Met Lys Ser Pro Ala Pro Ser Arg Pro
Gln Lys Met Ala Leu Ile Pro1 5 10 15Ala Cys Ile Phe Leu Cys Phe Ala
Ala Leu Ser Val Gln Ala Gln Val20 25 30Gln Leu
Lys354427PRTEscherichia coli 44Met Lys His Ser Val Ser Val Thr Cys
Cys Ala Leu Leu Val Ser Ser1 5 10 15Ile Ser Leu Ser Tyr Ala Ala Glu
Val Pro Ser20 254527PRTEscherichia coli 45Met Lys His Ser Val Ser
Val Thr Cys Cys Ala Leu Leu Val Ser Ser1 5 10 15Ile Ser Leu Ser Tyr
Ala Asp Ile Glu Leu Thr20 254627PRTEscherichia coli 46Met Lys His
Ser Val Ser Val Thr Cys Cys Ala Leu Leu Val Ser Ser1 5 10 15Ile Ser
Leu Ser Tyr Ala Gln Val Gln Leu Lys20 254725PRTEscherichia coli
47Met Lys Trp Leu Cys Ser Val Gly Ile Ala Val Ser Leu Ala Leu Gln1
5 10 15Pro Ala Leu Ala Asp Asp Leu Phe Gly20 254824PRTEscherichia
coli 48Met Lys Trp Leu Cys Ser Val Gly Ile Ala Val Ser Leu Ala Leu
Gln1 5 10 15Pro Ala Leu Asp Ile Glu Leu Thr204925PRTEscherichia
coli 49Met Lys Trp Leu Cys Ser Val Gly Ile Ala Val Ser Leu Ala Leu
Gln1 5 10 15Pro Ala Leu Ala Gln Val Gln Leu Lys20
255027PRTEscherichia coli 50Met Lys Lys Ser Ile Leu Ala Leu Ser Leu
Leu Val Gly Leu Ser Thr1 5 10 15Ala Ala Ser Ser Tyr Ala Ala Leu Pro
Glu Thr20 255127PRTEscherichia coli 51Met Lys Lys Ser Ile Leu Ala
Leu Ser Leu Leu Val Gly Leu Ser Thr1 5 10 15Ala Ala Ser Ser Tyr Ala
Asp Ile Glu Leu Thr20 255227PRTEscherichia coli 52Met Lys Lys Ser
Ile Leu Ala Leu Ser Leu Leu Val Gly Leu Ser Thr1 5 10 15Ala Ala Ser
Ser Tyr Ala Gln Val Gln Leu Lys20 255322PRTEscherichia coli 53Met
Lys Leu Ala Ala Cys Phe Leu Thr Leu Leu Pro Gly Phe Ala Val1 5 10
15Ala Ala Ser Trp Thr Ser205422PRTEscherichia coli 54Met Lys Leu
Ala Ala Cys Phe Leu Thr Leu Leu Pro Gly Phe Ala Val1 5 10 15Ala Asp
Ile Glu Leu Thr205522PRTEscherichia coli 55Met Lys Leu Ala Ala Cys
Phe Leu Thr Leu Leu Pro Gly Phe Ala Val1 5 10 15Ala Gln Val Gln Leu
Lys205627PRTEscherichia coli 56Met Lys Lys Leu Val Leu Ser Leu Ser
Leu Val Leu Ala Phe Ser Ser1 5 10 15Ala Thr Ala Ala Phe Ala Ala Ile
Pro Gln Asn20 255727PRTEscherichia coli 57Met Lys Lys Leu Val Leu
Ser Leu Ser Leu Val Leu Ala Phe Ser Ser1 5 10 15Ala Thr Ala Ala Phe
Ala Asp Ile Glu Leu Thr20 255827PRTEscherichia coli 58Met Lys Lys
Leu Val Leu Ser Leu Ser Leu Val Leu Ala Phe Ser Ser1 5 10 15Ala Thr
Ala Ala Phe Ala Gln Val Gln Leu Lys20 255928PRTEscherichia coli
59Met Lys Ile Lys Asn Ile Leu Leu Thr Leu Cys Thr Ser Leu Leu Leu1
5 10 15Thr Asn Val Ala Ala His Ala Lys Glu Val Lys Ile20
256028PRTEscherichia coli 60Met Lys Ile Lys Asn Ile Leu Leu Thr Leu
Cys Thr Ser Leu Leu Leu1 5 10 15Thr Asn Val Ala Ala His Ala Asp Ile
Glu Leu Thr20 256128PRTEscherichia coli 61Met Lys Ile Lys Asn Ile
Leu Leu Thr Leu Cys Thr Ser Leu Leu Leu1 5 10 15Thr Asn Val Ala Ala
His Ala Gln Val Gln Leu Lys20 256226PRTEscherichia coli 62Met Leu
Ala Phe Ile Arg Phe Leu Phe Ala Gly Leu Leu Leu Val Ile1 5 10 15Ser
His Ala Phe Ala Ala Thr Val Gln Asp20 256326PRTEscherichia coli
63Met Leu Ala Phe Ile Arg Phe Leu Phe Ala Gly Leu Leu Leu Val Ile1
5 10 15Ser His Ala Phe Ala Asp Ile Glu Leu Thr20
256426PRTEscherichia coli 64Met Leu Ala Phe Ile Arg Phe Leu Phe Ala
Gly Leu Leu Leu Val Ile1 5 10 15Ser His Ala Phe Ala Gln Val Gln Leu
Lys20 256514DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 65gagggcaaaa aatg
146616DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 66aggagaaaat aaaatg 166716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 67aggagatata catatg 1668236PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
68Met Met Ile Thr Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala1
5 10 15Gly
Val Met Ser Ala Gln Ala Met Ala Asp Ile Glu Leu Thr Gln Pro20 25
30Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Ser Cys Ser35
40 45Gly Asn Ala Leu Gly Asp Lys Tyr Ala Ser Trp Tyr Gln Gln Asn
Pro50 55 60Gly Gln Ala Pro Val Leu Val Ile Tyr Asp Asp Ser Asp Arg
Pro Ser65 70 75 80Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly
Asn Thr Ala Thr85 90 95Leu Thr Ile Ser Gly Thr Gln Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys100 105 110Gln Ser Tyr Asp Ser Pro Gln Val Val
Phe Gly Gly Gly Thr Lys Leu115 120 125Thr Val Leu Gly Gln Pro Lys
Ala Ala Pro Ser Val Thr Leu Phe Pro130 135 140Pro Ser Ser Glu Glu
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu145 150 155 160Ile Ser
Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp165 170
175Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys
Gln180 185 190Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu195 200 205Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln
Val Thr His Glu Gly210 215 220Ser Thr Val Glu Lys Thr Val Ala Pro
Thr Glu Ala225 230 23569249PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 69Met Lys Ile Lys Thr Gly
Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr1 5 10 15Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Gln Val Gln Leu Lys Glu20 25 30Ser Gly Pro Ala
Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys35 40 45Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ser Gly Val Gly Val Gly Trp50 55 60Ile Arg
Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala Leu Ile Asp65 70 75
80Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu Lys Thr Arg Leu Thr85
90 95Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr
Asn100 105 110Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg
Tyr Pro Val115 120 125Thr Gln Arg Ser Tyr Met Asp Val Trp Gly Gln
Gly Thr Leu Val Thr130 135 140Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Leu Pro Leu Ala Pro145 150 155 160Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val165 170 175Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala180 185 190Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly195 200
205Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly210 215 220Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys225 230 235 240Val Asp Lys Lys Val Glu Pro Lys
Ser2457027DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 70aaacatatgg atatcgaact gacccag
277127DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 71aaactgcagt tatcaggcct cagtcgg
277237DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 72aaactgcagg agatatacat atgcaggtgc aattgaa
377327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 73aaaaagcttt tatcagcttt tcggttc
277429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 74aaacatatga aatacctatt gcctacggc
297524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 75aaaaagctta ctacgaggag acgg 24
* * * * *
References