U.S. patent application number 10/643083 was filed with the patent office on 2004-08-05 for novel methods for obtaining, identifying and applying nucleic acid sequences and (poly)peptides which increase the expression yields of periplasmic proteins in functional form.
Invention is credited to Bothmann, Hendrick, Plueckthun, Andreas.
Application Number | 20040152103 10/643083 |
Document ID | / |
Family ID | 8227515 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152103 |
Kind Code |
A1 |
Plueckthun, Andreas ; et
al. |
August 5, 2004 |
Novel methods for obtaining, identifying and applying nucleic acid
sequences and (poly)peptides which increase the expression yields
of periplasmic proteins in functional form
Abstract
The present invention relates to a method for obtaining nucleic
acid sequences encoding (poly)peptides which increase the
expression yields of periplasmic proteins in functional form upon
co-expression of said (poly)peptides and said periplasmic proteins.
The invention also provides a method for the identification of said
(poly)peptides. Furthermore, the present invention relates to a
method for increasing the expression yields of periplasmic proteins
in functional form by co-expressing (poly)peptides, for example
Skp, FkpA, or a homolog of Skp or FkpA, in bacteria.
Inventors: |
Plueckthun, Andreas;
(Zurich, CH) ; Bothmann, Hendrick; (Zurich,
CH) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1666 K STREET,NW
SUITE 300
WASHINGTON
DC
20006
US
|
Family ID: |
8227515 |
Appl. No.: |
10/643083 |
Filed: |
August 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10643083 |
Aug 19, 2003 |
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09564351 |
May 1, 2000 |
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6630317 |
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Current U.S.
Class: |
435/5 ; 435/456;
702/20 |
Current CPC
Class: |
C07K 16/00 20130101;
C12N 15/1037 20130101; C07K 14/245 20130101; C40B 40/02 20130101;
C12Q 1/6811 20130101; C12N 15/70 20130101 |
Class at
Publication: |
435/006 ;
702/020; 435/456 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; C12N 015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 23, 1997 |
EP |
EP 97118457.7 |
Oct 23, 1998 |
WO |
PCT/EP98/06755 |
Claims
1. A method for obtaining a nucleic acid sequence comprising a
(poly)peptide coding sequence, which increases the expression yield
of a periplasmic protein in functional form in bacteria upon
co-expression of said periplasmic protein and said (poly)peptide,
comprising the steps of: (a) providing a collection of host cells
wherein each cell contains (i) a first nucleic acid sequence out of
a collection of nucleic acid sequences, and (ii) a second nucleic
acid sequence encoding said periplasmic protein; (b) causing or
allowing expression of (i) (poly)peptides expressible from said
collection of nucleic acid sequences, and (ii) said periplasmic
protein expressible from said second nucleic acid sequence; (c)
screening or selecting for a host cell expressing said periplasmic
protein with increased functional yield; (d) optionally, repeating
step (c) one or more times; (e) obtaining said first nucleic acid
sequence contained in said host cell.
2. The method of claim 1, further comprising the step of
identifying a (poly)peptide coding sequence comprised in said first
nucleic acid sequence.
3. The method of claims 1 or 2, wherein said first nucleic acid
sequence is or is derived from genomic DNA or mRNA of an organism,
or cDNA.
4. The method of anyone of claims 1 to 3, wherein said first
nucleic acid sequence comprises an at least partially randomized
sequence.
5. The method of anyone of claims 1 to 4, wherein (a) said first
nucleic acid sequence is comprised in a vector which can be
packaged in a filamentous phage particle, and (b) said periplasmic
protein is a fusion protein of at least part of a filamentous phage
coat protein and a further protein; and wherein in the course of
said expression a collection of filamentous phage particles
displaying said further protein is produced from said collection of
host cells.
6. The method of anyone of claims 1 to 5 wherein said further
protein comprises at least a domain of the immunoglobulin
superfamily, and preferably of the immunoglobulin family.
7. The method of claim 6 wherein said further protein is an
immunoglobulin fragment taken from the list of Fv, scFv,
disulphide-linked Fv, and Fab fragments.
8. A method for identifying a (poly)peptide which increases the
expression yield of a periplasmic protein in functional form in
bacteria upon co-expression of said periplasmic protein and said
(poly)peptide, comprising the steps of: (a) identifying a nucleic
acid sequence or a (poly)peptide coding sequence according to a
method of anyone of claims 1 to 7, and (b) deducing a (poly)peptide
therefrom.
9. A method for increasing the expression of a periplasmic protein
in functional form in a bacterial host cell, characterized by
co-expressing said periplasmic protein and a (poly)peptide
identified by the method according to claim 8.
10. The method of claim 9, wherein said periplasmic protein is a
member of a collection of periplasmic proteins expressed in a
collection of host cells.
11. The method of claims 9 or 10 wherein said (poly)peptide is the
E. coli protein Skp or a homolog thereof.
12. The method of claims 9 or 10 wherein said (poly)peptide is the
E. coli protein FkpA or a homolog thereof.
13. The method of anyone of claims 9 to 12 wherein said periplasmic
protein ia a fusion protein of at least part of a filamentous phage
coat protein and a further protein.
14. The method of anyone of claims 9 to 13 wherein said further
protein comprises at least a domain of the immunoglobulin
superfamily, and preferably of the immunoglobulin family.
15. The method of claim 14, wherein the further protein is an
immunoglobulin fragment taken from the list of Fv, scFv,
disulphide-linked Fv, and Fab fragment.
Description
[0001] The present invention relates to a method for obtaining
nucleic acid sequences encoding (poly)peptides which increase the
expression yields of periplasmic proteins in functional form upon
co-expression of said (poly)peptides and said periplasmic proteins.
The invention also provides a method for the identification of said
(poly)peptides. Furthermore, the present invention relates to a
method for increasing the expression yields of periplasmic proteins
in functional form by co-expressing (poly)peptides, for example
Skp, FkpA, or a homolog of Skp or FkpA, in bacteria.
[0002] Expression in the bacterial periplasm is the most convenient
route to express foreign recombinant proteins, especially proteins
containing disulphides, since the bacterial disulphide forming and
isomerization machinery (Bardwell, 1994) can be utilised.
Nevertheless, not all proteins can be produced with high functional
yield in the E. coli periplasm, and no general method for
optimizing the expression in functional form of poorly folding
proteins secreted into the periplasm exists.
[0003] Another field where the correct folding of proteins in the
periplasm is of crucial importance is in phage display. This method
has been used over the last decade to screen libraries not only of
peptides but also of a large variety of proteins (Dunn, 1996;
McGregor, 1996). These displayed proteins are fused to a phage coat
protein, e.g. to the N-terminus of the whole gene-3-protein (g3p)
or to its C-terminal domain. These proteins therefore fold in the
periplasm, while remaining anchored to the inner membrane by the
C-terminal hydrophobic extension of g3p, before being incorporated
into the phage coat. Therefore, the g3p fusion-proteins will almost
certainly fold in the same environment and use the same machinery
as periplasmically expressed proteins. Poorly folding proteins will
most likely be lost over multiple screening rounds irrespective of
their binding properties.
[0004] Co-expression of the cytoplasmic chaperonins GroEL and GroES
during M13 phage assembly for Fab display were reported to lead to
a 200fold increase in phage titer (Soderlind, 1993). However, the
relative amount of functional antibody fragments being displayed by
the phage particles was not affected. It was speculated that
GroEL/GroES assist in phage packing and assembly, although these
steps take place in the periplasm. A general method for increasing
the functional display of proteins on phage is not yet
available.
[0005] Consequently, there has been great interest in the question
of the existence of periplasmic chaperones. However, unlike the
well-characterized cytoplasmic machinery of E. coli, DnaK/DnaJ/GrpE
and GroEL/GroES and possibly others (Makrides, 1996; Martin &
Hartl, 1997; Buchner, 1996; EP 0 774 512 A3), the chaperone
composition of the periplasm has remained poorly understood (Wall
& Pluckthun, 1995; Missiakas et al., 1996). While progress in
elucidating the signal transduction of periplasmic stress has been
made (Missiakas & Raina, 1997), the ultimate effector molecules
controlling periplasmic folding have remained obscure, although
some proteins, such as FkpA or SurA, were believed to act as
general periplasmic folding catalysts (Missiakas et al., 1996).
FkpA has first been described as very similar to the eukaryotic
FK506 binding proteins (FKBPs) (Horne and Young, 1995), a class of
well-characterized peptidyl-prolyl cis-trans isomerases (PPIs),
which have been shown to be inhibited by the macrolipide FK506.
Missiakas and co-workers showed, that the mature FkpA is located in
the periplasm and assayed its activity (Missiakas et al., 1996).
The estimated Kcat/Km of the cis-trans isomeration of the Ala-Pro
peptidyl-prolyl bond using succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
as substrate was 90 mM-1s-1. FkpA is directly regulated by
.sigma..sup.E, which binds in its promoter region (Danese and
Silhavy, 1997). The .sigma..sup.E pathway is induced by heat stress
and conditions, that lead to misfolding or misassembly of outer
membrane proteins (OMPs), such as over-expression of OMPs or
inactivation of the surA gene.
[0006] Another protein which has been discussed in the context of
periplasmic folding and protein transport is Skp. Skp is a very
basic protein, which at first led to its misassignment as a
DNA-binding protein (Holck et al., 1987), later as an outer
membrane associated protein (Hirvas et al., 1990; Koski et al.,
1990; Koski et al., 1989), and a variety of synonyms (OmpH, HlpA)
witness its unclear function. Homologs have been found in
Salmonella typhimurium (Koski et al., 1990; Koski et al., 1989),
Yersinia enterocolitica (Hirvas et al., 1991), Yersinia
pseudotuberculosis (Vuorio et al., 1991), Haemophilus influenzae
(Fleischmann et al., 1995) and Pasteurella multocida (Delamarche et
al., 1995). Muller and co-workers (Thome et al., 1990) showed that
this protein stimulates the in vitro import of E. coli proteins
into membrane vesicles and subsequently established its periplasmic
location (Thome & Muller, 1991), consistent with its soluble
nature and the presence of a signal sequence. More recently, it was
proposed to be involved in the transport of outer membrane proteins
(Chen & Henning, 1996), and when its promoter region was
interrupted by a Tn10 transposon, the extreme heat shock factor
.sigma..sup.E (.sigma..sup.24) dependent response was induced
(Missiakas et al., 1996). However, it remained unclear whether this
is an effect of the absence of Skp or a polar effect on other
proteins located downstream of skp. The heat shock response was
probably induced indirectly via a change in the concentration of
outer membrane proteins, which is known (Missiakas et al., 1996) to
induce .sigma..sup.E (.sigma..sup.24).
[0007] However, attempts to increase the expression of antibody
fragments in functional form by over-expressing E. coli disulphide
isomerase DsbA and/or proline cis-trans isomerase PPlase A did not
significantly change the folding limit (Knappik et al., 1993). It
was concluded that aggregation steps in the periplasm compete with
periplasmic folding, and that they may occur before disulphide
formation and/or proline cis-trans isomerization take place and be
independent of their extent.
[0008] In summary, no protein has up to now been identified, which
unambiguously acts as a periplasmic chaperone and which could be
used to optimize the expression yield of a periplasmic protein in
functional form.
[0009] Thus, the technical problem underlying the present invention
is to identify factors which increase the expression yield of
periplasmic proteins in functional form in bacteria and to apply
these factors to the optimization of expression of periplasmic
proteins. The solution to the above technical problem is achieved
by providing the embodiments characterized in the claims.
Accordingly, the present invention allows to identify and to apply
nucleic acid sequences encoding (poly)peptides which increase the
expression yield of periplasmic proteins in functional form, and/or
to identify and apply the (poly)peptides. The technical approach of
the present invention, i.e. the co-expression of a collection of
(poly)peptides with said periplasmic protein in a collection of
host cells to screen or select for such nucleic acid sequences
and/or (poly)peptides is neither provided nor suggested by the
prior art.
[0010] Thus, the present invention relates to a method for
obtaining a nucleic acid sequence comprising a (poly)peptide coding
sequence, which increases the expression yield of a periplasmic
protein in functional form in bacteria upon co-expression of said
periplasmic protein and said (poly)peptide, comprising the steps
of:
[0011] (a) providing a collection of host cells wherein each cell
contains
[0012] (i) a first nucleic acid sequence out of a collection of
nucleic acid sequences, and
[0013] (ii) a second nucleic acid sequence encoding said
periplasmic protein;
[0014] (b) causing or allowing expression of
[0015] (i) (poly)peptides expressible from said collection of
nucleic acid sequences, and
[0016] (ii) said periplasmic protein expressible from said second
nucleic acid sequence;
[0017] (c) screening or selecting for a host cell expressing said
periplasmic protein with increased functional yield;
[0018] (d) optionally, repeating step (c) one or more times;
[0019] (e) obtaining said first nucleic acid sequence contained in
said host cell.
[0020] The term "obtaining a nucleic acid sequence" as used herein
includes the at least partial identification of the nucleic acid
molecule e.g. by sequencing and/or collecting the nucleic acid
molecules by biochemical techniques, for example, comprised in a
vector.
[0021] In the context of the present invention, the term
"(poly)peptide" relates to molecules consisting of one or more
chains of multiple, i. e. two or more, amino acids linked via
peptide bonds.
[0022] The term "protein" refers to (poly)peptides where at least
part of the (poly)peptide has or is able to acquire a defined
three-dimensional arrangement by forming secondary, tertiary, or
quaternary structures within and/or between its (poly)peptide
chain(s). This definition comprises proteins such as naturally
occurring or at least partially artificial proteins, as well as
fragments or domains of whole proteins, as long as these fragments
or domains have a defined three-dimensional arrangement as
described above.
[0023] The term "periplasmic protein" relates to proteins which,
after biosynthesis in the cytoplasm, are transported across the
inner membrane into the periplasm. This definition comprises
proteins which remain in soluble or associated form in the
periplasm, which are inserted in the inner or outer membrane, which
are further secreted into the medium or which are assembled into
complex structures such as filamentous phages particles which are
then secreted. The periplasmic proteins will normally, but not
necessarily, have at least a transport signal which directs the
protein to the periplasm.
[0024] The term "periplasmic protein in functional form" relates to
a periplasmic protein, which has a defined function, and which
folds during and after expression in a way which leads to a defined
three-dimensional arrangement required for the protein to be
functional. A "defined function" according to the present invention
is any feature of the protein which depends on the correctly folded
three-dimensional arrangement, and which can be detected or
determined. This comprises functions such as enzymatic activity or
binding to a target or binding partner, such as in the case of
receptor/ligand or antibody/antigen pairs. In addition, in the
context of the present invention, said "feature" referred to
hereinabove may be the presence of the correctly folded
three-dimensional arrangement itself, detected or determined, for
example, by an antibody recognizing the correctly assembled
three-dimensional arrangement of the protein, or by measuring
physico-chemical properties such as fluorescence or .alpha.-helix
content in fluorescence or CD spectra, respectively.
[0025] The term "expression yield of a periplasmic protein in
functional form" relates to the amount of a periplasmic protein
being produced in functional form on expression.
[0026] The term "(poly)peptides expressible from said first nucleic
acid sequences" relates to (poly)peptides for which open reading
frames (ORFs) exist on said first nucleic acid sequences and where
preferably the operator elements necessary for expression are
present on the corresponding vectors comprising said nucleic acid
sequences. In the case that said nucleic acid sequences comprise
fragments of genomic DNA, more than one ORF may be comprised in
anyone of said nucleic acid sequences.
[0027] The term "functional yield" relates to the amount of said
periplasmic protein being produced in functional form.
[0028] Methods of designing, creating or obtaining nucleic acid
sequences for expression, of constructing appropriate vectors,
inserting nucleic acid sequences into vectors, choosing appropriate
host cells, introducing vectors into host cells, causing or
allowing expression of (poly)peptides or protein, isolating nucleic
acids from host cells or identifying nucleic acid sequences and
corresponding protein sequences are standard methods (Sambrook et
al., 1989) which are well known to anyone of ordinary skill in the
art.
[0029] In a preferred embodiment, the method of the present
invention further comprises the step of identifying a (poly)peptide
coding sequence comprised in said first nucleic acid sequence.
[0030] The term "identifying a (poly)peptide coding sequence
comprised in said first nucleic acid sequence" relates to the
situation referred to hereinabove, where more than one ORF is
present in said first nucleic acid sequence. When more than one ORF
is found, the identifcation optionally further comprises the
analysis of individual ORFs and, if necessary, further testing such
as repeating steps (a) to (e) with a set of nucleic acid sequences
separately representing the individual ORFs. Said further testing
can be performed by anyone of ordinary skill in the art.
[0031] In a further preferred embodiment, said periplasmic protein
is not expressible, or in very low yields, in functional form when
expressed under standard conditions, i. e. without the
co-expression of said (poly)peptides.
[0032] In another embodiment, the present invention relates to a
method, wherein said periplasmic protein is a resistance marker, a
nutritional marker, a reporter protein, a transactivator of
transcription of marker genes or reporter genes, or a protein
binding to a target. As has been stated hereinabove in step (c),
the functional yield is determined by screening or selecting for an
increase in protein function.
[0033] If the protein is a periplasmic resistance marker such as
.beta.-lactamase or zeocin causing resistance to a certain
antibiotic when functionally present in the periplasm, a selection
is possible by culturing the host cells in the presence of said
antibiotic. Host cells expressing the marker in functional form
will be selected for.
[0034] If the protein is a periplasmic nutritional marker such as
maltose-binding protein or an amino-acid-binding protein, a
selection is possible by using auxotrophic host cells and by
culturing the cells in the presence of maltose, or the amino acid,
respectively. Host cells expressing the marker in functional from
will be selected for.
[0035] If the protein is a periplasmic reporter protein such as
alkaline phosphatase, a screening is possible by culturing the host
cells in the presence of the corresponding substrate resulting in a
colour reaction. Host cells expressing the reporter protein in
functional form will be selected for.
[0036] If the protein is a secreted protein having enzymatic
activity or binding to a target, a screening of the supernatant of
individual cell cultures or of a collection of host cells on a
plate can be performed by adding the appropriate substrate or
target, respectively, to the medium and measuring or determining
the amount of functional protein being secreted.
[0037] It will be possible for a person of ordinary skill in the
art, without undue burden, to identify and adapt existing screening
or selection protocols, e.g. based on the various ELISA formats
known, to arrive at protocols which are suitable for the indidual
proteins and the corresponding function to be screened of selected
for.
[0038] In a further preferred embodiment, said first nucleic acid
sequence is or is derived from genomic DNA or mRNA of an organism,
or cDNA.
[0039] Further preferred is a method, wherein said genomic DNA is
randomly fragmented. Genomic DNA can be fragmented by use of
restriction enzymes or DNA cleaving enzymes, chemical cleavage,
mechanical shearing or sonification. These are standard procedures
well known to anyone of ordinary skill in the art (Sambrook et al.,
1989).
[0040] In a yet further preferred embodiment of the present
invention, said first nucleic acid sequence comprises an at least
partially randomized sequence.
[0041] Such at least partially randomized sequences can be
generated in various ways well known to the practitioner in the
field, e.g. by random DNA syntheses using mixtures of
mononucleotides or trinucleotides (Virneks et al., 1994). There are
numerous examples of collections of nucleic acid sequences encoding
random peptide or antibody libraries which could be used in
accordance with the present invention.
[0042] In a further preferred embodiment, the present invention
relates to a method, wherein
[0043] (a) said first nucleic acid sequence is comprised in a
vector which can be packaged in a filamentous phage particle,
and
[0044] (b) said periplasmic protein is a fusion protein of at least
part of a filamentous phage coat protein and a further protein;
[0045] and wherein in the course of said expression a collection of
filamentous phage particles displaying said further protein is
produced from said collection of host cells.
[0046] The term "filamentous phage particles displaying said
further protein" refers to particles prepared by the phage display
method which has been developed and used extensively in the past 10
years. In said method, a foreign (poly)peptide or protein is
genetically fused to a coat protein of a phage, in most cases of a
filamentous phage such as M13, f1 of fd, whereby said phage
displays said foreign (poly)peptide or protein at its surface. Many
important aspects of phage display are summarized in various
publications (e.g. Kay et al., 1996).
[0047] In one further embodiment of the present invention, the
vector wherein said first nucleic acid sequence is comprised is a
phage vector or a phagemid vector. In the latter case, a helper
phage will be used to supply phage proteins not encoded on the
phagemid vector.
[0048] In another embodiment of the present invention, the phage
coat protein is the gVIp, gVIIIp or preferably gIIIp.
[0049] In a preferred embodiment of the present invention, binding
of the displayed protein to a cognate binding partner is screened
or selected for.
[0050] If the protein is an antibody, the cognate binding partner
is the corresponding antigen (and vice versa). In the case of a
receptor, the cognate binding partner is its ligand (and vice
versa).
[0051] The particular advantage of this embodiment of the method of
the present invention is that rare events leading to an increase in
functional yield can be selected for since the selected phage
particles can be used for infection of host cells and can thus be
amplified.
[0052] In yet another embodiment, said screening or selection is
for activity of the displayed further protein.
[0053] If the activity is an enzymatic activity, the supernatant of
individual host cell cultures can be used to assay for the
enzymatic activity.
[0054] In a still further embodiment, said further protein
comprises at least a domain of the immunoglobulin superfamily, and
preferably of the immunoglobulin family.
[0055] In the context of the present invention, the term
immunoglobulin superfamily (IgSF) refers to a family of proteins
which are characterized by having at least a domain with the
immunoglobulin fold, said superfamiliy comprising the
immunoglobulins or antibodies, and various other proteins such as
T-cell receptors or integrins.
[0056] In a most preferred embodiment, said further protein is an
immunoglobulin fragment taken from the list of Fv, scFv,
disulphide-linked Fv, and Fab fragments.
[0057] In this context, the term "Fv" refers to a fragment
comprising the VL (variable light) and VH (variable heavy) portions
of the antibody molecule, a "single-chain Fv" is a fragment, in
which the VL and VH chains are joined, in either a VL-VH, or VH-VL
orientation, by a peptide linker. A "disulphide-linked Fv" is a
fragment stabilized by an inter-domain disulphide bond. This is a
structure which can be made by engineering into each chain a single
cysteine residue, wherein said cysteine residues from two chains
become linked through oxidation to form a disulphide. The term
"Fab" refers to a complex comprising the VL-CL (variable and
constant light) and VH-CH1 (variable and first constant heavy)
portions of the antibody molecule.
[0058] In yet a further preferred embodiment, the invention relates
to the method wherein said first and second nucleic acid are
encoded on the same or on different vectors.
[0059] In a still further embodiment, the present invention relates
to a method for identifying a (poly)peptide which increases the
expression yield of a periplasmic protein in functional form in
bacteria upon co-expression of said periplasmic protein and said
(poly)peptide, comprising the steps of:
[0060] (a) identifying a nucleic acid sequence or a (poly)peptide
coding sequence according to a method of the invention as outlined
hereinabove, and
[0061] (b) deducing a (poly)peptide therefrom.
[0062] The deduction of a (poly)peptide can be achieved by
translating the (poly)peptide encoding sequence into an amino acid
sequence. By comparing the deduced (poly)peptide sequence with
published protein sequences, or by comparing the (poly)peptide
coding sequence identified as described above with published
nucleic acid sequences, larger (poly)peptides, or (poly)peptide
coding sequences, respectively, can be deduced and identified in
cases where said first nucleic acid sequence did not comprise the
full-length nucleic acid coding sequence of a protein. In addition
to the method described hereinabove, the (poly)peptide may be
identified directly by known methods from the host cells screened
or selected for. For example, said (poly)peptide may be expressed
as a fusion with a detection or labelling tag. The tagged
(poly)peptide may be isolated and identified by amino acid
sequencing.
[0063] In a most preferred embodiment, the present invention
relates to a method for increasing the expression of a periplasmic
protein in functional form in a bacterial host cell, characterized
by co-expressing said periplasmic protein and a (poly)peptide
identified by a method according to the the present invention.
[0064] Preferably, said bacterial host cells are E. coli cells.
[0065] In a further preferred embodiment, said periplasmic protein
is not expressible, or in very low yields, in functional form when
expressed under standard conditions, i. e. without the
co-expression of said (poly)peptides.
[0066] In a yet further preferred embodiment, said periplasmic
protein is a member of a collection of periplasmic proteins
expressed in a collection of host cells.
[0067] Several methods such as the phage display technology
referred to hereinabove provide libraries of proteins for screening
or selection procedures. However, the success of the procedures is
limited by differences in expression yields of functional library
members. For example, in the case antibody fragments, it is known
that the expression yields of fragments in functional form vary to
a large extent. A high percentage of fragments comprised in
antibody fragment libraries derived from immunoglobulin repertoires
is found not to be expressible, or in very low yield when expressed
under standard conditions, i. e. without the co-expression of said
(poly)peptides.
[0068] When expressing periplasmic proteins with yet unknown
biological function, or a collection of periplasmic proteins for
the identifcation or a member with a certain property (e. g. when
expressing an antibody fragment library with the goal to identify a
fragment which binds to a pre-defined target), the term "expression
. . . in functional form" refers to structural features rather than
to a defined biological function. In that context, a protein can be
called "functional" when it folds into a three-dimensional
arrangement representative for that kind of proteins. For example,
when expressing a collection of antibody molecules of fragments
thereof, a "expression . . . in functional form" is achieved when
said molecules of fragments are expressed in a correctly folded
form, the so-called immunoglobulin fold, since correct folding of
an antibody binding site is a prerequisite for its function, i.e.
the binding to a target.
[0069] In a further preferred embodiment, said (poly)peptide is the
E. coli protein Skp or a homolog thereof.
[0070] In a further preferred embodiment, said (poly)peptide is the
E. coli protein FkpA or a homolog thereof.
[0071] Proteins are termed homologous if the percentage of the sum
of identical and/or similar residues exceeds a defined threshold.
This threshold is commonly regarded by those skilled in the art as
being exceeded when at least 15% of the amino acids in the aligned
genes are identical, and at least 30% are similar. Similarity in
that context refers to the physico-chemical properties of the amino
acids, such as e.g. size, polarity, or charge.
[0072] Proteins which are homologous to Skp are known from
organisms such as Salmonella typhimurium (Koski et al., 1990; Koski
et al., 1989), Yersinia enterocolitica (Hirvas et al., 1991),
Yersinia pseudotuberculosis (Vuorio et al., 1991), Haemophilus
influenzae (Fleischmann et al., 1995) and Pasteurella multocida
(Delamarche et al., 1995).
[0073] Proteins which are homologous to FkpA are present e.g. in
many pathogenic bacteria (Horne and Young, 1995). In Legionella
pneumophila the corresponding protein showing PPI activity is
called MipA.
[0074] In a yet further preferred embodiment, the invention relates
to a method wherein said periplasmic protein is a fusion protein of
at least part of a filamentous phage coat protein and a further
protein.
[0075] Still further preferred is a method wherein said further
protein comprises at least a domain of the immunoglobulin
superfamily, and preferably of the immunoglobulin family.
[0076] Most preferably, the invention relates to a method wherein
the further protein is an immunoglobulin fragment taken from the
list of Fv, scFv, disulphide-linked Fv, and Fab fragment.
[0077] In yet a further preferred embodiment, the invention relates
to the method wherein the nucleic acid sequence encoding said
(poly)peptide, preferably Skp, FkpA, or a homolog of Skp or FkpA,
and the gene encoding said periplasmic protein are encoded on the
same or on different vectors, or wherein the nucleic acid sequence
encoding the (poly)peptide, preferably Skp, FkpA, or a homolog of
Skp or FkpA, is integrated in the genome of the bacterial host.
FIGURE CAPTIONS
[0078] FIG. 1. Selection scheme. A. Principle of selection. An E.
coli genomic library is co-expressed with a scFv-fragment, fused to
g3p. While the antibody is the same throughout, its folding yield
varies depending on the co-expressed factor. This factor is not
displayed on the phage, but expressed in the host cell producing
the phage, which in the case of an useful factor leads to better
"quality" scFv fragments displayed. Since the gene for the factor
is encoded on the phage, its information becomes enriched by phage
panning on antigen. B. Phagemid vector used for library
construction.
[0079] FIG. 2. Analysis of phagemid pools after different panning
rounds. For each round of phage proliferation, phagemids were
prepared from cells harvested from overnight cultures. The phagemid
pools were analyzed by restriction digest with Notl. M: Pstl
digested .lambda.-DNA as molecular weight marker, lane 1 to 7:
phagemids from infected cells after the panning round. lane 0:
phagemids before the first panning round.
[0080] FIG. 3. Schematic representation of the 952 bp insert
enriched by phage display and panning. Yaet is the product of an
ORF of 810 aa of unknown function. It shows 66.2% similarity to
amino acids of the protective surface antigen D15 of Haemophilus
influenzae and those of other bacteria (see Example 1.3.). The gene
lpxD codes for
UDP-3-O-[3-hydroxymyristoyl]-glucosamine-N-acyltransferase. The
only complete ORF found on this insert is the gene skp. Note that
the Sau3Al fragment obtained is the smallest one which contains the
full expression unit of Skp. SD, Shine-Dalgarno sequence; p,
promoter region, predicted by neural network analysis (Reese,
1994).
[0081] FIG. 4. Antigen-binding ELISAs of phages grown with or
without over-expressed Skp, displaying the scFv fragments 4-4-20
(Nieba et al., 1997), 4D5Flu (Jung and Pluckthun, 1997), FITC-E2
(Vaughan et al., 1996; Krebber et al., 1997a) and ABPC48-CH22S
(Proba et al., 1997; Proba et al.; 1998). Phages were purified by
CsCl gradients as described in Example 1.2. Phages grown in the
presence of over-expressed Skp reach higher antigen-binding ELISA
signals compared to phages grown without over-expressed Skp.
Inhibition with soluble antigen shows that binding is specific.
[0082] FIG. 5. Phage blot. Phage carrying g3p-fusion of the scFv
fragments 4-4-20 (Nieba et al., 1997), 4D5Flu (Jung and Pluckthun,
1997), FITC-E2 (Vaughan et al., 1996; Krebber et al., 1997a),
McPC603-H11 (Knappik and Pluckthun, 1995), ABPC48-CH22S (Proba et
al., 1997; Proba et al.; 1998) and AL214 were grown with or without
over-expressed Skp. Phages were purified by CsCl gradients as
described in Example 1.2. In the presence of over-expressed Skp,
more fusion protein is incorporated into the phages than in the
absence of Skp on the plasmid. Helper phage VCS M13 (Stratagene)
was loaded as size reference for g3p wt.
[0083] FIG. 6. Crude extract ELISA. From E. coli JM83 expressing
the soluble scFv fragments 4-4-20 (Nieba et al., 1997) and
ABPC48-CH22S (Proba et al., 1997; Proba et al.; 1998) with or
without over-expressed Skp crude extracts were prepared as
described in Example 1.2. ScFv fragments produced in the presence
of Skp give higher antigen binding ELISA signals than without skp
on the plasmid. Inhibition with soluble antigen shows that binding
is specific.
[0084] FIG. 7. Analysis of phagemid pools after different panning
rounds. For each round of phage proliferation, phagemids were
prepared from cells harvested from overnight cultures. The phagemid
pools were analyzed by restriction digest with Notl. M: Pstl
digested .lambda.-DNA as molecular weight marker, lane 1 to 7:
phagemids from infected cells after the panning round. lane 0:
phagemids before the first panning round.
[0085] FIG. 8. Schematic representations of the 1629 bp and 1987
inserts enriched by phage display and panning. YheO is a protein
with strong similarity to H. influenzae H10575. FkpA is a
peptidyl-prolyl cis-trans isomerase. SlyX is a protein with yet
unknown function. SlyD is a peptidyl-prolyl cis-trans isomerase.
YheP is a protein with yet unknown function.
[0086] FIG. 9. Antigen-binding ELISAs of phages grown with or
without over-expressed Skp, FkpA, SlyX, FkpA+SlyX, and FkpA+Skp,
displaying the scFv ABPC48-C(H22)S (Proba et al., 1997; Proba et
al., 1998). Phages were purified by CsCl gradients as described in
Example 1.2. Phages grown in the presence of over-expressed Skp
reach higher antigen-binding ELISA signals compared to phages grown
without over-expressed Skp. Inhibition with soluble antigen shows
that binding is specific.
[0087] The examples illustrate the invention:
EXAMPLES
Example 1
Identification and Application of Skp
[0088] 1.1. Introduction
[0089] We have made use of the hypothesis, that g3p fusion-proteins
might fold in the same environment and use the same machinery as
periplasmically expressed proteins, in a search for cellular
factors which might aid both the folding of periplasmic proteins
and proteins displayed on phage. We have used a very poorly folding
single-chain Fv fragment of the antibody 4-4-20 (Bedzyk et al.,
1990), (Whitlow et al., 1995), specific for fluorescein, as a model
system. Previous work (Nieba et al., 1997) showed that this scFv
strongly aggregates in the bacterial periplasm, even though the
same protein, once in the native state, is very soluble and stable.
This indicates that not the final product limits the yield, but
that the folding pathway branches off to aggregates. For this and
similar cases, the folding yield is a kinetic and not a
thermodynamic problem, and can thus potentially be helped by
cellular factors.
[0090] We wished to identify such factors without any prejudice
concerning whether they are membrane-bound, periplasmic, or even
cytoplasmic proteins. We also wanted to be able to find any factors
with might affect the total yield of the product without directly
influencing folding. We therefore developed a selection system
making use of phage display (FIG. 1A). The poorly folding scFv
fragment was displayed as a fusion protein with g3p, and a library
of E. coli proteins was co-expressed on the same phagemid. We
reasoned that, if a particular E. coli cell produces a beneficial
factor encoded on this phagemid, this cell will give rise to phages
which outcompete the other phages, because a higher fraction of the
phages will display correctly folded scFv. Thus, while the
displayed scFv is genetically identical on all phages, this method
selects for the effect of the additional factor encoded on the same
phagemid, even though the factor itself is not displayed. This
factor improves the "quality" of the scFv, namely the percentage of
correctly folded molecules displayed on the phage, by acting on the
host cell which produces the particular phage.
[0091] Using this strategy (FIG. 1A), a gene from E. coli was
enriched, coding for the periplasmic protein Skp, which had been
suspected previously to have a role in folding or transport of
outer membrane proteins (Chen & Henning, 1996).
[0092] 1.2. Experimental Protocols
[0093] Construction of genomic library. A Notl site was inserted in
the phage display vector pAK100 (Krebber et al., 1997) at position
5656. A polylinker was inserted as an oligonucleotide cassette into
this Notl site. The gel-purified Sfil fragment encoding the scFv
fragment of the anti-fluorescein antibody 4-4-20 (Bedzyk et al.,
1990) was ligated in this vector pHB100, yielding the plasmid
pHB102. Genomic DNA of E. coli JM83 was isolated with Qiagen-tip
100G according to the manufacturer's protocol. The genomic DNA was
partially digested with Sau3Al and applied to a 1% agarose gel. The
range of 1 kb to 6 kb length was cut out, and the genomic DNA
eluted with GenElute.TM. agarose spin columns (Supelco),
phenol/chloroform extracted and ethanol precipitated. After
ligation of the E. coli library in the Bglll site of the polylinker
of pHB102, the ligation mixture was precipitated with n-butanol and
electroporated into E. coli XL1-Blue (Stratagene). After plating on
2.times.YT in 530 cm.sup.2 dishes (Nunc) and overnight incubation
at 37.degree. C., the colonies were washed off the plates with 5 ml
2.times.YT, the OD.sub.550 was determined and the cells stored at
-80.degree. C. after addition of glycerol to 10% final
concentration.
[0094] Phage panning. A 10 ml culture of 2.times.YT, containing 15
.mu.g/ml tetracycline (tet), containing 0.1 ml salt mixture (0.86 M
NaCl, 0.25 M KCl, 1 M MgCl.sub.2) was inoculated to an OD.sub.550
of 0.1 with E. coli harboring the genomic library. After 1 h
incubation at 37.degree. C. chloramphenicol (cam) was added to a
concentration of 30 .mu.g/ml, and the cells were grown to an
OD.sub.550 of 0.5. Then 10.sup.12 pfu of helper phage VCS M13
(Stratagene) was added and incubated for 15 min without agitation
at 37.degree. C., followed by addition of 50 ml 2.times.YT medium
containing 30 .mu.g/ml cam, 15 .mu.g/ml tet, 0.5 ml salt mixture,
0.1 mM isopropyl-.beta.-D-thiogalactoside (IPTG), and then shaken
for 2 h at 37.degree. C. After addition of 30 .mu.g/ml kanamycin
(kan) the cultures were grown overnight at 37.degree. C. The cells
were harvested and the phagemid DNA isolated (QIAprep spin kit,
Qiagen). The phages from the culture supernatant were precipitated
by incubation for 30 min with 1/4 volume PEG/NaCl solution (17% PEG
6000, 3.3 M NaCl, 1 mM EDTA) on ice, and the pellets were
redissolved in 2 ml PBS (8 mM Na.sub.2HPO.sub.4, 1.8 mM
KH.sub.2PO.sub.4, 137 mM NaCl, 3 mM KCl, pH 7.4 (Sambrook et al.,
1989)). Immunotubes (Nunc) were coated with 20 .mu.g/ml
fluorescein-isothiocyanate coupled to bovine serum albumin
(FITC-BSA) in PBS overnight at 4.degree. C. and blocked with 5%
skimmed milk in PBST (PBS containing 0.05% Tween-20) for at least 1
h at room temperature. Five hundred .mu.l of the phage solution was
filled to a final volume of 5 ml with 2% skimmed milk in PBST and
applied to the tubes for 2 h at room temperature. The tubes were
washed 20 times with PBST and 2 times with PBS. Bound phages were
eluted with 1 ml 0.1 M glycine/HCl pH 2.2 for 10 min. The eluate
was neutralized immediately with 60 .mu.l 2 M Tris and the phages
(typically 10.sup.4-10.sup.6 cfu) were used for re-infection.
[0095] Phage purification and ELISA. Phage ELISAs were carried out
to assay the amount of functionally displayed scFv on M13 phages.
Single colonies were grown at 37.degree. C. overnight in 5 ml
2.times.YT medium containing 30 .mu.g/ml cam and 15 .mu.g/ml tet.
Ten ml of 2.times.YT medium containing 30 .mu.g/ml cam, 15 .mu.g/ml
tet, 0.4% glucose and 0.1 ml salt mixture was inoculated with the
overnight culture to give an OD.sub.550 of 0.1. At an OD.sub.550 of
0.3 to 0.5, 10.sup.12 cfu VCS helper phage (Stratagene) were added.
After 15 min, 50 ml 2.times.YT medium containing 30 .mu.g/ml cam,
15 .mu.g/ml tet, 0.5 ml salt mixture and 0.1 mM IPTG was added.
After 2 h at 37.degree. C., kan was added to a final concentration
of 30 .mu.g/ml and the cells were grown overnight. The phages were
precipitated from the culture supernatant by incubating for 30 min
with 1/4 volume PEG/NaCl solution as above on ice, and the pellets
were redissolved in 1 ml PBS.
[0096] After addition of 1.6 g of CsCl, the volume was adjusted to
4 ml with PBS. The CsCl solution was transferred into a
1/2.times.11/2 in. polyallomer tube and centrifuged at 100,000 rpm
for 4 hr in a TLN-100 rotor (Beckman Intruments) at 4.degree. C.
After centrifugation the phage band was removed as described (Smith
& Scott, 1990). The phages were transferred to 1/2.times.2 in.
polycarbonate tubes, which were filled with PBS to 3 ml. After
centrifugation at 50,000 rpm for 1 hr in a TLA-100.3 rotor at
4.degree. C., the pelleted phages were redissolved in 3 ml PBS.
After an additional centrifugation at 50,000 rpm for 1 hr in a
TLA-100.3 rotor at 4.degree. C., the phages were dissolved in PBS.
The concentration of phage particles was quantified
spectrophotometrically (Day, 1969).
[0097] ELISA plates were coated with FITC-BSA in PBS, for
anti-levan antibodies with 10 .mu.g/ml levan (polyfructose, Sigma)
in PBS at 4.degree. C. overnight. The plates were blocked for 1 h
at room temperature. A defined number of purified phages (measured
by OD) (Day, 1969) were mixed with 2% skimmed milk in PBST in the
absence or presence of 10 .mu.M fluorescein or 0.05% levan and
applied to the blocked ELISA plates and incubated for 1 h at room
temperature. Detection was as above, using an anti-M13 antibody
conjugated with horseradish peroxidase (Pharmacia).
[0098] Phage blots. For phage blots 10.sup.11 phages were applied
to a reducing 11% SDS-PAGE, and blotted on nitrocellulose
membranes. Detection was carried out with the monoclonal antibody
10C3 (Tesar et al., 1995), which recognizes the C-terminal half of
g3p (1:50.000 in TBST (25 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.05%
Tween-20) containing 2% milk), for 60 min at RT, followed by
incubation with a polyclonal anti-mouse-peroxidase conjugate
(Pierce) (1:5000 in TBST/2% milk, 45 min RT), and using the ECL-kit
(Amersham).
[0099] Crude extract ELISA. Fifty ml of LB medium containing 30
.mu.g/ml cam were inoculated with a single colony of E. coli JM83,
harboring a plasmid encoding the respective scFv fragment. The
cultures were grown at 24.degree. C. to an OD.sub.550 of 0.5 and
induced with 1 mM IPTG. After overnight induction at 24.degree. C.
the cells were harvested and resuspended in 4 ml PBS. Whole cell
extracts were prepared by French Press lysis at 10,000 psi and 1 ml
of the crude extract was centrifuged in an Eppendorf tube for 30
min at 50.000 rpm in a TLA-100.3 rotor (Beckman Instruments) at
4.degree. C. After centrifugation the supernatants containing the
soluble material were normalized to an OD.sub.550 of 20 in 1 ml.
ELISA plates were coated and blocked as described above for phage
ELISAs. A defined amount of crude extract was mixed with 2% skimmed
milk in PBST in the absence or presence of 10 .mu.M fluorescein or
0.05% levan and applied to the blocked ELISA plates and incubated
for 1 h at room temperature. The signal was detected with an
anti-myc-tag antibody (Munro & Pelham, 1986) and an anti-mouse
antibody conjugated with horseradish peroxidase and soluble BM blue
POD-substrate (Boehringer-Mannheim), and after stopping the
reaction with 0.1 M HCl, the signals were read at 405 nm.
[0100] Protein purification. The anti-phosphorylcholine scFv
McPC603-H11 (Knappik & Pluckthun, 1995) was purified using
PC-Sepharose affinity chromatography (Skerra & Pluckthun, 1988)
in the presence or absence of co-expressed Skp. The concentration
and yield was estimated photometrically using a calculated
extinction coefficient (Gill & von Hippel, 1989).
[0101] 1.3. Identification of Skp
[0102] We used a phagemid displaying the poorly folding scFv
fragment of the anti-fluorescein antibody 4-4-20 (Bedzyk et al.,
1990; Nieba et al., 1997) as the recipient for an E. coli genomic
library. E. coli DNA was size-fractionated from 1 to 6 kb and
ligated into a polylinker placed between the colE1 origin of
replication and the chloramphenicol (cam) resistance gene of a
phagemid (FIG. 1B) developed for phage display (Krebber et al.,
1997). Thus, E. coli genes, regulated under their own promoters,
are over-expressed on the phagemid, primarily through an effect of
vector copy number. A library size of 5.times.10.sup.4 clones
ensured that each piece of the E. coli genome should be
represented, provided it led to viable clones.
[0103] Seven panning rounds on BSA-fluorescein were carried out
(FIG. 1A), and after each round the phagemid DNA was cut with the
restriction enzyme Notl to detect the accumulation of any inserts.
It can be seen in FIG. 2 that a band of about 990 bp accumulates
throughout the panning. Four of 8 single colonies analyzed after
the seventh round carried this insert, which was sequenced and
identified to only contain the complete gene for the periplasmic
protein Skp (Holck & Kleppe, 1988), from 272 bases upstream of
the start codon to 199 bases downstream of the stop codon (FIG. 3).
Four bases after the stop codon of skp is the start codon of lpxD
(firA), leading to a truncated peptide of the 65 N-terminal amino
acids of this protein. One-hundred and twenty-five base pairs
upstream of the start codon of the skp gene lies the stop codon of
an open reading frame, which codes for an 810 aa protein with
unknown function. A homology search showed 66.2% similarity to the
protective surface antigen D15 of Haemophilus influenzae
(Swiss-Prot: P46024), and surface proteins of Pasteurella multocida
(TREMBL: Q51930) and Neisseria gonorrhoeae (TREMBL: P95359).
[0104] 1.4. Effects of Skp Co-Expression on Phage Display
[0105] To determine how and why Skp gets enriched, we first
characterized the phages produced in the absence or presence of
co-expressed Skp. For this purpose, we cloned a variety of
different scFv fragments in the phagemid with and without skp. The
phage titer was indistinguishable within experimental error,
demonstrating that Skp is not selected because it would lead to the
production of more phages. However, the antigen binding phage ELISA
signal from the same number of purified phage particles is higher,
proving that the Skp over-expression increases the number of
functional antibody molecules on the phage (FIG. 4). This effect is
seen with all four antibodies tested, albeit to different
degrees.
[0106] We then determined whether the total amount of fusion
protein per phage is also increased by the over-expression of Skp.
For this purpose we analyzed the amount of full-length fusion
protein on purified phage particles in the presence or absence of
skp on the phagemid by Western blot, using the monoclonal antibody
10C3 (Tesar et al., 1995) (FIG. 5). For the scFv 4-4-20, the
co-expression of Skp dramatically increased the presence of fusion
protein presented on the phage. Since the same amount of purified
phages was loaded on the gel, Skp must facilitate the incorporation
of functional fusion protein into the phage, which is also
reflected by the antigen-binding ELISA (see above). It can be seen
that Skp has this effect on all of the six antibodies tested,
albeit to a different extent and to different final level of
incorporation. Since the fusion protein is still only a minor
species when compared to g3p wt (encoded by the helper phage), we
cannot reliably determine a decrease of g3p wt, but most likely,
the scFv-g3p fusion protein takes the place of g3p wt more often in
the presence of over-expressed Skp.
[0107] 1.5. Effects of Skp Co-Expression on Soluble Protein
Expression
[0108] We then determined the effect of Skp on the production of
several of the scFv fragments in soluble form using the
non-suppressor strain JM83. Using antigen-binding ELISA (FIG. 6) it
can be seen that the amount of soluble scFv was dramatically
increased in the presence of co-expressed Skp. To demonstrate that
this is also reflected in the yield of purified protein, the scFv
fragment of the anti-phosphorylcholine binding antibody McPC603-H11
(Knappik & Pluckthun, 1995) was tested. Co-expression of Skp
increased the amount of protein, purified by
affinity-chromatography on phosphorylcholine by about a factor of
4.
[0109] The results of FIG. 4 and FIG. 5 suggest that the more an
scFv fragment tends to aggregate in the periplasm of E. coli, the
stronger is the influence of Skp in the phage ELISA. For the scFv
fragment of FITC-E2 (Vaughan et al., 1996; Krebber et al., 1997a),
which folds very well und shows little insoluble material when
expressed in the periplasm, we observed a reduced influence of Skp
in the phage ELISA, compared to the poorly folding scFv 4-4-20
(Nieba et al., 1997) and ABPC48-C(H22)S (Proba et al., 1997; Proba
et al., 1998). The engineered Flu4D5 (Jung & Pluckthun, 1997),
with improved properties compared to 4-4-20, is intermediate. This
shows that the better an scFv is functionally expressed and the
less aggregation-prone it is, the less is the influence of Skp,
suggesting that Skp supports the correct folding of poorly
expressed scFv fragments and its fusion proteins.
Example 2
Identification and Application of FkpA
[0110] 2.1. Experimental Protocols
[0111] Construction of genomic library. The gel-purified Sfil
fragment encoding the scFv fragment of the anti-levan antibody
ABPC-C(H22)S (Proba et al., 1997; Proba et al., 1998) was ligated
in the vector pHB100 (Bothmann and Pluckthun, 1998), yielding the
plasmid pHB121. Genomic DNA of E. coli RC354c (Chen and Henning,
1996) was isolated with Nucleobond AXG100 cartridge according to
the manufacturer's protocol (Macherey-Nagel). The genomic DNA was
partially digested with Sau3Al and applied to a 1% agarose gel. The
range of 1 kb to 6 kb length was cut out, and the genomic DNA
eluted with GenElute.TM. agarose spin columns (Supelco),
phenol/chloroform extracted and ethanol precipitated. After
ligation of the E. coli library in the Bglll site of the polylinker
of pHB121, the ligation mixture was precipitated with n-butanol and
electroporated into E. coli XL1-Blue (Stratagene). After plating on
2.times.YT in 530 cm.sup.2 dishes (Nunc) and overnight incubation
at 37.degree. C., the colonies were washed off the plates with 5 ml
2.times.YT, the OD.sub.550 was determined and the cells stored at
-80.degree. C. after addition of glycerol to 50% final
concentration.
[0112] Phage panning. Phage panning was done as described in
Example 1. Instead using FITC-BSA, the immunotubes (Nunc) were
coated with 10 .mu.g/ml levan (polyfructose, Sigma) in PBS
overnight at 4.degree. C.
[0113] Phage purification and ELISA. Phage purification and ELISA
was done exactly as described in Example 1.
[0114] 2.2. Phage Selection and Identification of Co-Expressed
Factor.
[0115] We used a phagemid displaying the poorly folding scFv
fragment of the anti-levan antibody ABPC48-C(H22)S as the recipient
for an E. coli genomic library. E. coli RC354c-DNA (skp-deficient
strain) was size-fractionated from 1 to 6 kb and ligated into the
polylinker of plasmid pHB121. Thus, E. coli genes, regulated under
their own promoters, are over-expressed on the phagemid, primarily
through an effect of vector copy number. A library size of
6.4.times.10.sup.5 clones ensured that each piece of the E. coli
genome should be represented, provided it led to viable clones.
[0116] Seven panning rounds on levan were carried out, and after
each round the phagemid DNA was cut with the restriction enzyme
Notl to detect the accumulation of any inserts. It can be seen in
FIG. 7 that two bands of about 1.7 kb and 2.0 kb accumulate
throughout the panning. Fourteen of 17 single colonies analyzed
after the seventh round carried the 1.7 kb insert, 3 the 2.0 kb
insert. Both inserts were sequenced and the 1.7 kb band contains an
insert of 1629 bp length, the 2.0 kb band an insert of 1987 bp
length (FIG. 8). Both inserts contain the same two complete genes
coding for the periplasmic protein FkpA (Horne and Young, 1995;
Missiakas et al., 1996) and the protein SlyX with unknown function.
Both inserts start 218 bp upstream of the stop codon of fkpA.
Therefore both inserts code for the first 21 aa of the gene yheO,
which codes for a protein with strong similarity to Haemophilus
influenzae HI0575. The 1629 bp insert ends 159 bp downstream of the
stop codon of slyX, whereas the 1987 bp insert ends 528 bp
downstream of the stop codon of slyX. Both inserts code for the
C-terminal part of SlyD (Roof et al., 1994; Roof et al., 1997;
Hottenrott et al., 1997). The 1987 bp insert codes also for the
first 117 aa of YheP, an protein with unknown function. To examine,
which of the two complete genes is responsible for enrichment, fkpA
and slyX were PCR-amplified and precisely recloned separately as
well as in combination at the same position in the vector pHB102
(see Example 1).
[0117] 2.3. Characterization of the Influence of FkpA and SlyX on
Phage Display.
[0118] To determine how and why FkpA and SlyX get enriched, we
characterized the phages produced in the absence and presence of
co-expressed FkpA and SlyX. The antigen binding phage ELISA (FIG.
9) shows, that over-expression of FkpA increases the number of
functional antibody molecules on the phage, compared to no
over-expression or over-expression of Skp. In contrast, the
over-expression of SlyX has no effect on the number of functional
antibody molecules on the phage.
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