U.S. patent application number 11/504937 was filed with the patent office on 2006-12-21 for process for production of polypeptides.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Eriks Sasha Paegle, Dorothea Reilly, Daniel G. Yansura.
Application Number | 20060286640 11/504937 |
Document ID | / |
Family ID | 23047941 |
Filed Date | 2006-12-21 |
United States Patent
Application |
20060286640 |
Kind Code |
A1 |
Paegle; Eriks Sasha ; et
al. |
December 21, 2006 |
Process for production of polypeptides
Abstract
Vectors for producing polypeptides heterologous to prokaryotes
are described comprising, along with the polypeptide-encoding
nucleic acid, anti-termination nucleic acid that inhibits
intragenic transcription termination with a non-lambda promoter
therefor and/or nucleic acid encoding a GreA or GreB protein and a
promoter therefor. Also described are processes for producing a
heterologous polypeptide in prokaryotic host cells utilizing such
elements to improve the quality and/or quantity of heterologous
polypeptide produced.
Inventors: |
Paegle; Eriks Sasha;
(Seattle, WA) ; Reilly; Dorothea; (San Francisco,
CA) ; Yansura; Daniel G.; (Pacifica, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
|
Family ID: |
23047941 |
Appl. No.: |
11/504937 |
Filed: |
August 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11339922 |
Jan 26, 2006 |
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11504937 |
Aug 16, 2006 |
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10080866 |
Feb 22, 2002 |
7029876 |
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11339922 |
Jan 26, 2006 |
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60274384 |
Mar 9, 2001 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/471; 530/399; 536/23.5 |
Current CPC
Class: |
C12N 15/71 20130101;
A61K 39/0258 20130101; C12P 21/02 20130101; C07K 14/245 20130101;
Y02A 50/30 20180101; Y02A 50/474 20180101; C12N 15/70 20130101 |
Class at
Publication: |
435/069.1 ;
435/471; 435/252.3; 530/399; 536/023.5 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C12N 15/74 20060101
C12N015/74; C07K 14/50 20060101 C07K014/50; C07K 14/475 20060101
C07K014/475 |
Claims
1. A vector comprising nucleic acid encoding GreA or GreB protein,
nucleic acid encoding a polypeptide heterologous to prokaryotic
cells, and one or more promoters for the nucleic acids.
2. The vector of claim 1 wherein the nucleic acid encodes GreB.
3. The vector of claim 1 wherein the prokaryotic cells are
bacterial cells.
4. The vector of claim 1 wherein the polypeptide is a mammalian
polypeptide.
5. A process for producing a heterologous polypeptide in
prokaryotic host cells comprising: (a) culturing the host cells,
which comprise nucleic acid encoding GreA or GreB protein, nucleic
acid encoding the heterologous polypeptide, and one or more
promoters for the nucleic acids; and (b) recovering the
heterologous polypeptide from the cells or from cell culture
medium.
6. The process of claim 5 wherein nucleic acid encoding GreB
protein is expressed.
7. The process of claim 5 wherein the cells are bacterial
cells.
8. The process of claim 5 wherein the heterologous polypeptide is a
mammalian polypeptide.
9. The process of claim 5 wherein the mammalian polypeptide is a
human polypeptide.
10. The process of claim 9 wherein the human polypeptide is
thrombopoietin (TPO) or fibroblast growth factor-5 (FGF-5).
11. The process of claim 5 wherein the promoter is a trp or
alkaline phosphatase promoter or both.
12. The process of claim 5 wherein the polypeptide is recovered
from the cytoplasm or periplasm of the cells.
13. The process of claim 5 wherein the polypeptide is recovered
from the cell culture medium.
Description
RELATED APPLICATIONS
[0001] This is a divisional application claiming priority to
application Ser. No. 11/339,922, filed Jan. 26, 2006, which is a
continuation application claiming priority to application Ser. No.
10/080,866, filed Feb. 22, 2002, which is a non-provisional
application filed under 37 CFR 1.53(b)(1), claiming priority under
35 USC 119(e) to provisional application No. 60/274,384 filed Mar.
9, 2001, the contents and entire disclosure of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to improved vectors and methods for
producing polypeptides using such vectors. In particular, this
invention is related to improved expression of polypeptides from
nucleic acids such as cloned genes and production of various
polypeptides and proteins, including those of eukaryotic origin in
prokaryotic hosts.
[0004] 2. Description of Related Art
[0005] The level of production of a protein in a host cell is
governed by three major factors: the number of copies of its gene
within the cell, the efficiency with which those gene copies are
transcribed, and the efficiency with which the resultant messenger
RNA (mRNA) is translated. The quality of protein produced is
similarly governed by various factors, including the
anti-termination mechanism in the host cell.
[0006] Recombinant proteins produced in E. coli occasionally
contain structural modifications that restrict their usefulness as
therapeutic drugs or reagents for structure-function relationship
studies. Such modifications include N- and C-terminal truncations,
extensions, incomplete removal of N-terminal initiator methionine,
misincorporation of lysine for arginine, and norleucine for
methionine. For example, during the purification of recombinant
murine interleukin-6 from E. coli, it was observed that 5-10% of
the mIL-6 molecules contained a novel C-terminal modification (Tu
et al., J. Biol. Chem., 270: 9322-9326 (1995)).
[0007] This C-terminal "tag" is encoded by a small metabolically
stable RNA of E. coli (10Sa RNA) (Chauhan and Apirion, Mol.
Microbiol., 3: 1481-1485 (1989)). 10Sa RNA, also known as
transfer-messenger RNA, or tmRNA, contains a tRNA-like structure in
vivo with the 5'- and 3'-end sequences and an internal reading
frame encoding a "tag" peptide.
[0008] The primary cause of the production of truncated 10Sa-tagged
proteins is the translation of mRNA truncated within the coding
region (Keiler et al., Science, 271: 990-993 (1996)). Premature
transcription termination and RNase cleavage appear to be the major
factors capable of producing such truncated mRNA. The first of
these factors, premature transcription termination, is potentially
amenable to some type of transcription anti-termination. Several of
these systems have been described, including .lamda.N, .lamda.Q,
HK022, rrn, and Psu (Weisberg et al., J. Bacteriol., 181: 359-367
(1999)). Most of these systems are used to control gene expression
temporally in phage development by transcribing through intergenic
transcription terminators. The function of the rrn anti-termination
is somewhat different, and it has been proposed to prevent
rho-dependent transcription termination within the non-translated
ribosomal RNA operons.
[0009] Despite the accumulation of considerable knowledge of these
systems over many years, their only demonstrated usefulness in
terms of recombinant technology has been in the control of gene
expression by overriding intergenic transcriptional terminators
(Mertens et al., Bio/Technol., 13: 175-179 (1995)). Other reports
describe the failure of one of these systems (rrn) to alleviate
problems within a translated coding sequence containing extensive
secondary structure (Makrides, Microbiol. Rev., 60: 512-538
(1996)).
[0010] Several fusions of a protein with at least a portion of an
anti-terminator protein have been disclosed, especially the N gene
protein and most particularly the N-terminal fragment thereof (JP
9059299 published Mar. 4, 1997; WO 89/03886 published May 5, 1989;
WO 88/06628 published Sep. 7, 1988; U.S. Pat. No. 5,834,184 issued
Nov. 10, 1998; EP 700,997 published Mar. 13, 1996; U.S. Pat. No.
5,354,846 issued Oct. 11, 1994; U.S. Pat. No. 5,618,715 issued Apr.
8, 1997; U.S. Pat. No. 5,374,520 issued Dec. 20, 1994; Zhukovskaya
et al., Nucl. Acids. Res., 20: 6081-6090 (1992); Horiuchi et al.,
Biotechnol. Lett., 16: 113-118 (1994); Kamasawa et al., IFAC Symp.
Ser., 10: 255-258 (1992); Kovgan et al., Vopr. Virusol., 31:
485-489 (1986)).
[0011] Several plasmids that contain an N utilization site for
binding anti-terminator N protein produced by the host cell, such
as E. coli, have been constructed (U.S. Pat. No. 5,256,546 issued
Oct. 26, 1993; EP 691,406 published Jan. 10, 1996; U.S. Pat. No.
5,162,217 issued Nov. 10, 1992; EP 131,843 published Jan. 23,
1985). Other plasmids involving the N gene, operon, or portion
thereof have been described (SU 1405313 published Mar. 15, 1994; EP
314,184 published May 3, 1989; U.S. Pat. No. 4,578,355 issued Mar.
25, 1986; WO 85/04418 published Oct. 10, 1985; Rees et al., Proc.
Natl. Acad. Sci. USA, 93: 342-346 (1996).; Hwang et al., Biochem.
Biophys. Res. Commun., 173: 711-717 (1990); Bielawski et al., Acta
Biochim. Pol., 34: 29-34 (1987); Stanssens et al., Cell, 44:
711-718 (1986); Gatenby and Castleton, Mol. Gen. Genet., 185:
424-429 (1982)); Martin-Gallardo et al., J. Gen. Virol., 74:
453-458 (1993); Das, 72.sup.nd Annual Meeting of the American
Society of Biological Chemists, May 31-Jun. 4, 1981, Fed. Proc., 40
(6): 1764 (1981); Beck et al., Bio/Technology, 6: 930-935
(1988)).
[0012] The expression of gamma-interferon was found to increase
over two-fold when the .lamda.N anti-termination system was
eliminated and only the P.sub.L promoter was used (WO 85/02624
published Jun. 20, 1985). Cloning and expression vectors in which
the active N gene is preferably absent are also described (U.S.
Pat. No. 5,401,658 issued Mar. 28, 1995).
[0013] Transcription of DNA is often arrested at sites in DNA that
trap a fraction of elongating RNA polymerase molecules that pass
through, resulting in locked ternary complexes that cannot
propagate or dissociate their RNA product. Transcript cleavage
factors cleave the RNA in such complexes at the 3' end, allowing
RNA polymerase to back up and re-attempt to read through the
potential trap. In addition to assuring efficient transcript
elongation, transcript cleavage factors increase the fidelity of
transcription, since misincorporated bases at the 3' end of the
nascent RNA also lead to arrested complexes (Erie et al., Science,
262: 867-873 (1993)). Further, such factors allow RNA polymerase to
transcribe through strong blocks to elongation that can otherwise
arrest the enzyme on the DNA (Lee et al., J. Biol. Chem., 269:
22295-22303 (1994)). In addition, these factors can facilitate the
transition of RNA from the stage of abortive initiation to
elongation at certain promoters (Hsu et al., Proc. Natl. Acad. Sci.
USA, 92: 11588-11592 (1995)).
[0014] Both bacteria and eukaryotes contain proteins that can
stimulate such cleavage (Surratt et al., Proc. Natl. Acad. Sci.
USA, 88: 7983-7987 (1991); Borukhov et al., Proc. Natl. Acad. Sci.
USA, 89: 8899-8902 (1992); Borukhov et al., Cell, 72: 459-466
(1993); Izban and Luse, Genes & Dev., 6: 1342-1356 (1992);
Izban and Luse, J. Biol. Chem., 267: 13647-13655 (1992); Izban and
Luse, J. Biol. Chem., 268: 12864-12873 (1993); Izban and Luse, J.
Biol. Chem., 268: 12874-12885 (1993); Kassavetis and Geiduschek,
Science, 259: 944-945 (1993); Reines, J. Biol. Chem., 267:
3795-3800 (1992); Wang and Hawley, Proc. Natl. Acad. Sci. USA, 90:
843-847 (1993); Gu et al., J. Biol. Chem., 268: 25604-25616 (1993);
Guo and Price, J. Biol. Chem., 268: 18762-18770 (1993)). Two modes
of cleavage have been described. One yields one to three nucleotide
fragments and the other produces larger fragments, up to at least
12 nucleotides in size. Two transcript cleavage factors, GreA and
GreB, have been identified in E. coli (Borukhov et al., Proc. Natl.
Acad. Sci. USA, supra, and Borukhov et al., Cell, supra,
respectively). GreA-dependent transcript cleavage usually results
in the removal of di- and trinucleotides from the 3' end of the
stalled RNA. GreB-dependent cleavage yields larger
oligonucleotides, up to a length of nine nucleotides. Both proteins
bind RNA polymerase. Neither the GreA nor GreB proteins possess
intrinsic nuclease activity; rather, they stimulate a nuclease
activity inherent in RNA polymerase (Oriova et al., Proc. Natl.
Acad. Sci. USA, 92: 4596-4600 (1995)). The GreA and GreB proteins
are homologous, sharing 38% sequence identity and 59% sequence
similarity. It was found that GreA-induced transcript cleavage in
transcription complexes containing E. coli RNA polymerase is
controlled by multiple factors, including nascent transcript
location and structure (Feng et al., J. Biol. Chem., 269:
22282-22294 (1994)).
[0015] Crystallization of GreA has been disclosed (Darst et al., J.
Mol. Biol., 242: 582-585 (1994)) as well as its crystal structure
(Stebbins et al., Nature, 373: 636-640 (1995)). The organization
and functions of domains of GreA and/or GreB have been investigated
(Koulich et al., J. Biol. Chem., 272: 7201-7210 (1997); Koulich et
al., J. Mol. Biol., 276: 379-389 (1998); Polyakov et al., J. Mol.
Biol., 281: 465-473 (1998)). Moreover, purification and assay
procedures for GreA and GreB are reported (Borukhov and Goldfarb,
Meth. Enzymol., 274: 315-326 (1996)). Interactions between RNA
polymerase and transcript affect GreA- and GreB-mediated reverse
translocation (Feng et al., J. Cellular Biochem. Suppl., 0: 18C, p.
58 (1994)). Both GreA and GreB have been shown to enhance promoter
escape (Hsu et al., Proc. Natl. Acad. Sci. USA, 92: 11588-11592
(1995)).
[0016] In eukaryotes, the transcription elongation factor TFIIS,
otherwise known as SII (Reines et al., J. Biol. Chem., 264:
10799-10809 (1989); Sluder et al., J. Biol. Chem., 264: 8963-8969
(1989)), is similar to the GreA and GreB proteins in that it
stimulates RNA cleavage from the 3' end of RNA in a stalled complex
but does not share significant sequence homology with the GreA and
GreB protein (Borukhov et al., Cell, supra). TFIIS stimulates
either small or large fragment cleavage, depending on reaction
conditions and the particular complex examined (Izban and Luse, J.
Biol. Chem., 268: 12874-12885 (1993), supra; Wang and Hawley,
supra). Evidence for functional similarity between prokaryotic and
eukaryotic transcription elongation and read-through mechanisms has
been found (Mote and Reines, J. Biol. Chem., 273: 16843-16852
(1998)).
[0017] Homologs of E. coli GreA have been identified. The predicted
amino acid sequence encoded by the Rickettsia prowazekii greA gene
has 50.3% amino acid identity and 66.9% amino acid similarity to E.
coli GreA (Marks and Wood, Nucl. Acids Res., 20: 3785 (1992)). The
deduced amino acid sequence of GreA from Pseudomonas aeruginosa
exhibits 65.2% identity to its counterpart in E. coli K-12 (Lu et
al., J. Bacteriol., 179: 3043-3046 (1997)). Streptococcus
pneumoniae polypeptide GreA has also been disclosed, along with
methods of producing the GreA polypeptide by recombinant means and
for utilizing GreA or its antagonists for the treatment or
diagnosis of infection (EP 838,525 published Apr. 2, 1998).
Further, GreA from Staphylococcus aureus has also been-disclosed,
as well as recombinant methods of making it and methods for
utilizing it to screen for antibacterial compounds (EP 893,502
published Jan. 27, 1999).
[0018] There is a current and continuing need in the art for
improving the quality of recombinant protein produced by host cells
such that production of truncated forms of the protein, such as
10Sa-tagged material, is minimized or eliminated. There is also a
need for higher amounts of full-length protein produced by
prokaryotes.
SUMMARY OF THE INVENTION
[0019] Accordingly, the present invention provides, in one aspect,
a vector for producing a polypeptide heterologous to prokaryotic
cells comprising (1) anti-termination nucleic acid that inhibits
intragenic transcription termination with a non-lambda promoter
therefor, and (2) RNA encoding the polypeptide with a non-lambda
promoter therefor, wherein an RNA recognition site for binding
anti-termination protein produced from the nucleic acid is located
5' of the RNA encoding the polypeptide. Preferably, the vector
further comprises nucleic acid encoding a GreA or GreB protein with
a promoter therefor.
[0020] In another aspect, the invention provides a process for
producing a heterologous polypeptide in prokaryotic host cells
comprising:
[0021] (a) culturing the host cells, which comprise (1)
anti-termination nucleic acid that inhibits intragenic
transcription termination with a non-lambda promoter therefor, and
(2) RNA encoding the polypeptide with a non-lambda promoter
therefor, wherein an RNA recognition site for binding
anti-termination protein produced from the nucleic acid is located
5' of the RNA encoding the polypeptide, and wherein the
anti-termination nucleic acid is expressed at the time of
expression of the RNA; and
[0022] (b) recovering the heterologous polypeptide from the cells
or from cell culture medium.
[0023] The invention supplies, in yet another aspect, a vector
comprising nucleic acid encoding GreA or GreB protein and nucleic
acid encoding a polypeptide heterologous to prokaryotic cells,
preferably with one or more promoters for the nucleic acids.
[0024] In a still further aspect, the invention entails a process
for producing a heterologous polypeptide in prokaryotic host cells
comprising:
[0025] (a) culturing the host cells, which comprise nucleic acid
encoding GreA or GreB protein and nucleic acid encoding the
heterologous polypeptide, and one or more promoters for the nucleic
acids; and
[0026] (b) recovering the heterologous polypeptide from the cells
or from cell culture medium.
[0027] Lambda phage uses N anti-termination to control gene
expression by transcribing through strategically placed intergenic
terminators. The anti-termination system herein is found to be
effective against intragenic termination signals within
heterologous genes.
[0028] Moreover, the general trend in the literature over the years
is to eliminate the lambda N anti-termination system that was used
with the adjacent P.sub.L promoter system and just use the P.sub.L
promoter or replace the N gene with a polylinker or other fusion
partner. In contrast, the invention herein lies in using the
anti-termination system without the P.sub.L promoter.
[0029] Further, the system herein is designed specifically to
prevent the formation and accumulation of truncated and 10Sa-tagged
heterologous proteins, which cause problems with protein
purification. One of the primary causes of truncated and
10Sa-tagged proteins is premature transcription termination within
a translated coding sequence. The accumulation of full-length
protein may be similar with or without the anti-termination system,
but the accumulation of the truncated forms is significantly
reduced by promoting transcriptional readthrough of intragenic
termination signals within the protein's coding sequence.
[0030] Additionally, unwanted cleavage of the polypeptide is
minimized by inclusion of nucleic acids encoding GreA or GreB.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows the construction of plasmid pMP331.
[0032] FIG. 2 shows the construction of plasmid pMP843.
[0033] FIG. 3 shows the construction of plasmid pMP871.
[0034] FIG. 4 shows the construction of plasmid pMP931.
[0035] FIG. 5 shows the construction of plasmid pMP945.
[0036] FIG. 6 shows the construction of plasmid pMP951.
[0037] FIG. 7 shows the construction of plasmid pMP982.
[0038] FIG. 8 shows the construction of plasmid pMP1016.
[0039] FIG. 9 shows the construction of plasmid pMP1086.
[0040] FIG. 10 shows the construction of plasmid pMP1099.
[0041] FIG. 11 shows the construction of plasmid pMP1201.
[0042] FIG. 12 shows the construction of plasmid pMP1217.
[0043] FIG. 13 shows the construction of plasmid pJJ142.
[0044] FIG. 14 shows the construction of plasmid pDR1.
[0045] FIG. 15 shows the construction of plasmid pDR3.
[0046] FIGS. 16A-16C show an analysis of thrombopoietin (TPO)
expression under the transcriptional control of the tryptophan
(trp) and alkaline phosphatase (AP or phoA) promoters. On the left
are molecular weight markers in kDs. Lanes 1 are the negative
control plasmid pBR322 after induction of the trp promoter in M9
media, lanes 2 are the trp TPO expression plasmid pMP331 after
induction of the trp promoter, lanes 3 are the negative control
plasmid pBR322 after induction of the AP promoter in C.R.A.P.
media, and lanes 4 are the AP TPO expression plasmid pMP1099 after
induction of the AP promoter. FIG. 16A is a Coomassie blue-stained
SDS gel of whole cell extracts; FIG. 16B is a
histidine/horse-radish-peroxidase (HRP)-probed blot of whole cell
extracts (the whole cell lysate is separated by SDS-PAGE,
transferred to nitrocellulose, and probed with an agent that binds
to the polyhis motif on the TPO leader); and FIG. 16C is an
anti-10Sa polyclonal antibody Western blot of whole cell extracts
from TPO induction cultures. The arrows point to full-length
TPO.
[0047] FIGS. 17A and 17B respectively show the insertion of a nutL
site into the trp (SEQ ID NO:1) and AP (SEQ ID NO:2) TPO expression
constructs. In the FIG. 17A sequence, the nutL sequence was
inserted at the beginning of the mRNA sequence based on the
promoter -10 box. The FIG. 17B sequence shows the insertion of the
nutL site into the AP promoter construct. Here, the nut site is
situated further downstream from the mRNA start site.
[0048] FIGS. 18A and 18B show the expression of .lamda.N protein
under the transcriptional control of the tacII promoter. The start
sequence of the .lamda.N protein is shown in each of FIGS. 18A and
18B (SEQ ID NO:3). The FIG. 18A nucleotide sequence (SEQ ID NO:4)
shows the fusion of the tacII promoter to the .lamda.N coding
sequence. In this case the tacII Shine-Dalgarno sequence has been
deleted to provide for reduced .lamda.N translation, and an
alternative sequence with lower 16S ribosomal RNA binding is used.
The FIG. 18B nucleotide sequence (SEQ ID NO:5) shows the fusion of
the tacII promoter to the .lamda.N gene using the complete
Shine-Dalgarno sequence for high-level N expression.
[0049] FIGS. 19A-19C show an analysis of TPO expression with the
trp promoter +/- .lamda.N anti-termination. On the left are
molecular weight markers in kDs. Lanes 1 are the negative control
plasmid pBR322, lanes 2 are the trp TPO expression plasmid pMP331,
lanes 3 are the trp TPO expression plasmid pMP951 with .lamda.N
anti-termination and low-level N expression, and lanes 4 are the
trp TPO expression plasmid pMP1217 with .lamda.N anti-termination
and the high-level expression of N. FIG. 19A is a Coomassie
blue-stained SDS gel of whole cell extracts; FIG. 19B is a
histidine/horse-radish-peroxidase (HRP)-probed blot of whole cell
extracts; and FIG. 19C is an anti-10Sa polyclonal Western blot of
whole cell extracts. The arrows point to full-length TPO.
[0050] FIGS. 20A-20C show an analysis of TPO expression with the AP
promoter +/- .lamda.N anti-termination. On the left are molecular
weight markers in kDs. Lanes 1 are the negative control plasmid
pBR322, lanes 2 are the AP TPO expression plasmid pMP1099, lanes 3
are the AP TPO expression plasmid pMP1086 with .lamda.N
anti-termination and the low-level N expression, and lanes 4 are
the AP TPO expression plasmid pMP1201 with .lamda.N
anti-termination and the high-level expression of N. FIG. 20A is a
Coomassie blue-stained SDS gel of whole cell extracts; FIG. 20B is
a histidine/horse-radish-peroxidase (HRP)-probed blot of whole cell
extracts; and FIG. 20C is an anti-10Sa polyclonal antibody Western
blot of whole cell extracts. The arrows point to full-length
TPO.
[0051] FIG. 21 shows the construction of plasmid pFGF5IT.
[0052] FIG. 22 shows the construction of plasmid pFGF5IT-AT.
[0053] FIGS. 23A-23C show an analysis of FGF-5 expression +/-
.lamda.N anti-termination. Lanes 1 are the negative control plasmid
pBR322, lanes 2 are the expression of FGF-5 without .lamda.N
anti-termination (pFGF5IT), and lanes 3 are the expression of FGF-5
with .lamda.N anti-termination (pFGF5IT-AT). FIG. 23A is a
Coomassie blue-stained SDS gel of whole cell lysates from induced
fermentation cultures (equivalent O.D..sub.600); FIG. 23B is a
Western blot of whole cell lysates probed with an anti-FGF-5
antibody; and FIG. 23C is a Western blot of whole cell lysates
probed with an anti-10Sa antibody. The arrows point to full-length
FGF-5.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] As used herein, "greA" and "greB" refer to genes encoding
the transcript cleavage factors known in the literature as GreA and
GreB proteins, respectively, and amino acid sequence variants
thereof that are functional and effective for the same purpose of
transcript cleavage useful in the invention disclosed herein. They
may be from any source, with one example being the greA and greB
genes from E. coli as described by Borukhov et al., Proc. Natl.
Acad. Sci. USA, supra, and Borukhov et al., Cell, supra, and
another example being the greA genes disclosed by EP 838,525.
[0055] As used herein, "anti-termination factor" is an
anti-terminator protein that generally has RNA binding activity and
anti-terminator activity. Examples include the anti-terminator N
proteins of phages .lamda., .phi.21, and P22, which have been
completely sequenced. See Franklin, J. Mol. Biol., 181: 85-91
(1985) and Lazinski et al., Cell, 59: 207-218 (1989). All of these
N proteins contain an arginine-rich domain corresponding to about
amino acids 1-19 at the N-terminus of the protein that is
responsible for RNA binding activity of these proteins, while the
remainder of each protein confers anti-terminator activity
(Franklin, J. Mol. Biol., 231: 343-360 (1993)). Preferably, the
anti-termination factor is a phage factor. More preferably it is a
.lamda.N or .lamda.Q gene, more preferably a phage .lamda.N
protein.
[0056] An "RNA recognition site" refers to a site on an RNA
molecule that recognizes a specific protein. For example, an RNA
recognition site for binding anti-termination protein would be nut
for the N gene, and qut for the Q gene.
[0057] A "transcriptional terminator" or "transcriptional
termination signal" is operationally defined as a point where the
rate of release of an RNA transcript is greater than the rate of
addition of the next nucleotide. For purposes herein, the
terminator may be rho-dependent or rho-independent. An "intragenic"
terminator is one that is homologous to the heterologous
polypeptide coding sequence herein. An "intergenic" terminator is
one that is exogenous to the heterologous polypeptide coding
sequence herein, for example, bacterial or bacteriophage
termination signals when the polypeptide is mammalian in
origin.
[0058] "Anti-termination nucleic acid that inhibits intragenic
transcription termination" signifies nucleic acid encoding
anti-termination factors that block or override intragenic
transcriptional terminators within heterologous genes. This
definition includes the N and Q genes, as well as rrn and HK022
anti-termination nucleic acid. Preferably, it is the N or Q
gene.
[0059] As used herein, "cistron" is a distinctly translatable
sequence defined by having a single messenger RNA transcript with
one promoter.
[0060] As used herein, "polycistronic" refers to a polynucleotide
comprising two or more cistrons where several different genes are
transcribed as a single message from their operons, and two or more
pairs of start and stop codons.
[0061] As used herein, "polypeptide", or "polypeptide of interest"
refers generally to peptides and proteins-having more than about
ten amino acids. The polypeptides are "heterologous," i.e., foreign
to the host cell being utilized, such as a human protein produced
by a bacterial cell, or a bacterial polypeptide produced from a
bacterial cell line that is not the native source of the
polypeptide. Preferably, the polypeptide is mammalian, and most
preferably human.
[0062] Examples of mammalian polypeptides include molecules such
as, e.g., renin, a growth hormone, including human growth hormone;
bovine growth hormone; growth hormone releasing factor; parathyroid
hormone; thyroid stimulating hormone; lipoproteins;
.alpha.1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;
thrombopoietin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; clotting factors such as factor
VIIIC, factor IX, tissue factor, and von Willebrands factor;
anti-clotting factors such as Protein C; atrial naturietic factor;
lung surfactant; a plasminogen activator, such as urokinase or
human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha
and -beta; enkephalinase; a serum albumin such as human serum
albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin
B-chain; prorelaxin; mouse gonadotropin-associated peptide; a
microbial protein, such as beta-lactamase; DNASE; inhibin; activin;
vascular endothelial growth factor (VEGF); receptors for hormones
or growth factors; integrin; protein A or D; rheumatoid factors; a
neurotrophic factor such as brain-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-.alpha.; cardiotrophins
(cardiac hypertrophy factor) such as cardiotrophin-1 (CT-1);
platelet-derived growth factor (PDGF); fibroblast growth factors
such as aFGF, bFGF, and FGF-5; epidermal growth factor (EGF);
transforming growth factor (TGF) such as TGF-alpha and TGF-beta,
including TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or
TGF-.beta.5; insulin-like growth factor-I and -II (IGF-I and
IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor
binding proteins; CD proteins such as CD-3, CD-4, CD-8, CD-19,
CD-20, and CD-40; erythropoietin; osteoinductive factors;
immunotoxins; a bone morphogenetic protein (BMP); an interferon
such as interferon-alpha, -beta, and -gamma; colony stimulating
factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs),
e.g., IL-1 to IL-10; anti-HER-2 antibody; superoxide dismutase;
T-cell receptors; surface membrane proteins; decay accelerating
factor; viral antigen such as, for example, a portion of the AIDS
envelope; transport proteins; homing receptors; addressins;
regulatory proteins; antibodies; and fragments of any of the
above-listed polypeptides.
[0063] Preferred mammalian polypeptides include t-PA, gp120,
anti-HER-2, anti-CD20, anti-CD11a, anti-CD18, anti-CD40, DNase,
IGF-I, IGF-II, FGF-5, thrombopoietin, brain IGF-I, thrombopoietin,
growth hormone, relaxin chains, growth hormone releasing factor,
insulin chains or pro-insulin, urokinase, immunotoxins,
neurotrophins, and antigens. Particularly preferred mammalian
polypeptides include, e.g., t-PA, gp120 (IIIb), anti-HER-2,
anti-CD20, anti-CD11a, anti-CD18, anti-CD40, DNase, thrombopoietin,
IGF-I, IGF-II, FGF-5, growth hormone, NGF, NT-3, NT-4, NT-5, and
NT-6, and most preferably, FGF-5 and thrombopoietin.
[0064] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes include a promoter such as the AP or trp
promoter, optionally an operator sequence, and a ribosome-binding
site.
[0065] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is
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.
[0066] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0067] A "nut" site refers to the N-utilization site to which the N
protein binds. The term includes both natural nut and manipulations
or variations thereof that still work with the N protein by binding
thereto to effect mRNA-specific lambda N anti-termination. Examples
include lambda nutL, nutR, Box B by itself, Box A and Box B, mutant
nut sites, and nut sites from related lambdoid phages. These are
described, for example, in Friedman et al., Genes Dev., 4:
2210-2222 (1990); Mogridge et al., J. Biol. Chem., 273: 4143-4148
(1998); Olson et al., Cell, 31: 61-70 (1982); Patterson et al., J.
Mol. Biol., 236: 217-228 (1994); and Schauer et al., J. Mol. Biol.,
194: 679-690 (1987).
[0068] A "non-lambda promoter" and "non-lambda" termination sites
indicate respectively promoters and termination sites not from
lambda phage, for example, not the lambda P.sub.L promoter.
Examples of suitable non-lambda promoters herein include, for
example, the trp and AP promoters.
B. MODES FOR CARRYING OUT THE INVENTION
[0069] In one aspect, a vector is provided for producing a
polypeptide heterologous to prokaryotic cells, preferably a
mammalian polypeptide, and most preferably a human polypeptide.
Such vector has at least the following elements: anti-termination
nucleic acid that inhibits intragenic transcription termination,
RNA encoding the polypeptide, and one or separate non-lambda
promoters for the nucleic acid and RNA. In this vector an RNA
recognition site for binding the anti-termination protein produced
from the nucleic acid is located 5' of the RNA encoding the
polypeptide. The vector may further include nucleic acid encoding a
GreA or GreB protein along with a promoter for this nucleic acid,
which can be lambda or non-lambda.
[0070] In another aspect, a process is described for producing a
heterologous polypeptide in prokaryotic host cells. In this process
the host cells are cultured and the polypeptide is recovered from
the cells or cell culture medium. The cells comprise the components
of the above-described vector, i.e., anti-termination nucleic acid
that inhibits intragenic transcription termination and RNA encoding
the polypeptide, with non-lambda promoter(s) for each, wherein an
RNA recognition site for binding the anti-termination protein
produced from the nucleic acid is located 5' of the RNA encoding
the polypeptide. In this process the anti-termination nucleic acid
is expressed at the time of expression of the RNA.
[0071] In another embodiment, a vector is set forth that includes
nucleic acid encoding GreA or GreB protein and nucleic acid
encoding a heterologous polypeptide, along with one or more
promoters therefor, which may be lambda or non-lambda
promoters.
[0072] In a still further embodiment, a process for producing a
heterologous polypeptide in prokaryotic host cells is provided
involving culturing the host cells, which comprise the components
of the above-described GreA/GreB vector, i.e., nucleic acid
encoding GreA or GreB protein and nucleic acid encoding the
heterologous polypeptide, and one or more promoters for the nucleic
acids. After the culturing step, the heterologous polypeptide is
recovered from the cells or from the cell culture medium.
[0073] The anti-termination system may be performed with or without
the presence of nucleic acid encoding GreA or GreB, since by itself
the anti-termination system described above results in a
significant decrease in heterologous protein truncation and 10Sa
tagging and leads to a corresponding increase in full-length
protein. The co-expression with GreA or GreB nucleic acid leads to
more recombinant protein production, regardless of whether the
anti-termination system described herein is used.
[0074] The anti-termination nucleic acid as well as the GreA/GreB
nucleic acid and the nucleic acid encoding the heterologous protein
may be cDNA or genomic DNA from any source. The anti-termination
and GreA/GreB nucleic acids are generally the native sequence, but
need not be if they provide the same benefits to heterologous
polypeptide production as set forth herein.
[0075] If the anti-termination factors or GreA or GreB proteins are
native products of the host cell, and if the factors controlling
expression of these native genes are understood, such factors can
be manipulated to achieve over-expression of these genes, e.g., by
induction of transcription from the natural promoter using known
inducer molecules, by mutation of the nucleic acids controlling or
repressing expression of the gene product to produce a mutant
strain that inductively over-expresses the gene product, by second
site mutations which depress the synthesis or function of factors
that normally repress the transcription of the gene product, and
the like.
[0076] The heterologous nucleic acid (e.g., cDNA or genomic DNA) is
suitably inserted into a replicable vector for expression in the
prokaryotic cells under the control of a suitable promoter for
prokaryotic cells. Many vectors are available for this purpose, and
selection of the appropriate vector will depend mainly on the size
of the nucleic acid to be inserted into the vector and the
particular host cell to be transformed with the vector. Each vector
contains various components depending on its function
(amplification of DNA or expression of DNA) and the particular host
cell with which it is compatible. The vector components for
prokaryotic cell transformation generally include, but are not
limited to, one or more of the following: a signal sequence, an
origin of replication, one or more marker genes, and a
promoter.
[0077] The promoters herein may be constitutive or inducible,
preferably inducible, and are recognized by the host prokaryotic
organism and operably linked to the GreA/GreB-, and/or
anti-termination-, and polypeptide-encoding nucleic acid components
of the vectors herein. For the anti-termination plasmid, the
promoter is non-lambda. For the plasmid that does not contain
anti-termination nucleic acid, the promoter may be lambda or
non-lambda. The vectors herein contain either one promoter for all
two or three elements, provided it is appropriate for all the
elements, or two or more separate promoters, which may be the same
or different provided they are appropriate, operably linked to each
of the nucleic acids encoding the anti-termination factor, the
polypeptide, and/or GreA/GreB protein. The promoters are selected
to be compatible with the cell type in which expression is to be
performed.
[0078] For the anti-termination factor the promoter is non-lambda,
and for the GreA/GreB the promoter may be lambda or non-lambda.
Suitable non-lambda promoters for use in the preferred cell type,
E. coli, include, for example, the .beta.-lactamase and lactose
(lac) promoter systems (Chang et al., Nature, 275: 615 (1978);
Goeddel et al., Nature, 281: 544 (1979)), the arabinose promoter
system (Guzman et al., J. Bacteriol., 174: 7716-7728 (1992)), AP, a
trp promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980)
and EP 36,776), hybrid promoters such as the tac promoter (deBoer
et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)), and the T3
or T7 promoter (See generally, e.g., Itakura et al., Science, 198:
1056-1063 (1977); Goeddel et al., Proc. Natl. Acad. Sci. USA, 76:
106-110 (1979), Emtage et al., Nature, 283: 171-174 (1980); and
Martial et al., Science, 205: 602-606 (1979)). The most preferred
non-lambda promoters herein are the trp and AP promoters.
[0079] Suitable lambda or non-lambda promoters for use with
prokaryotic hosts include the non-lambda promoters set forth above,
plus the lambda promoters, i.e., those from lambda phage, for
example, the lambda P.sub.L promoter and the lambda P.sub.r
promoter. However, other known lambda and non-lambda promoters are
suitable. Their nucleotide sequences have been published, thereby
enabling a skilled worker operably to ligate them to DNA encoding
the polypeptide of interest or to the anti-termination or GreA or
GreB genes (Siebenlist et al., Cell, 20: 269 (1980)) using linkers
or adaptors to supply any required restriction sites.
[0080] Promoters for use in prokaryotic systems also generally
contain a Shine-Dalgarno (SD) sequence operably linked to the DNA
encoding the polypeptide of interest. The promoter can be removed
from the prokaryotic source DNA by restriction enzyme digestion and
inserted into the vector containing the desired DNA.
[0081] If the host contains a pstS variant gene, the expression
vector for producing a heterologous polypeptide suitably contains
an AP promoter that is recognized by the host organism and is
operably linked to the nucleic acid encoding the polypeptide of
interest. This promoter initiates increased levels of transcription
from DNA under its control in response to a decreased concentration
of inorganic phosphate in the culture medium. The AP promoter can
be removed from the prokaryotic source DNA by restriction enzyme
digestion and inserted into the vector containing the desired
DNA.
[0082] In one alternative, the prokaryotic cells comprise two
separate vectors respectively containing the anti-termination
nucleic acid and/or GreA/GreB protein and the RNA encoding the
heterologous polypeptide.
[0083] In another alternative, the anti-termination or greA/greB
nucleic acid and the RNA encoding the heterologous polypeptide are
contained on the same vector and are under the control of a single
promoter or more than one separate inducible promoters. In this
case, the polypeptide may be suitably fused in-frame to an
anti-terminator protein and/or GreA or GreB protein as defined
above such that the combined coding sequence is operably linked to
a single promoter. Hence, the polypeptide gene and the
anti-termination nucleic acid and/or greA/greB nucleic acid are
suitably coupled to form a polycistronic unit. Alternatively, they
may be independently expressed under separate, differently
inducible promoters on the same vector so that initiation of
expression can occur in the proper order.
[0084] The anti-termination and/or greA/greB nucleic acid and
polypeptide nucleic acid can be anywhere in the cell cytoplasm,
including the chromosome. Hence, they may be integrated into the
host cell genome or contained on autonomously replicating plasmids.
The anti-termination nucleic acid such as the N or Q gene can be
expressed with any reasonably controlled promoter, and is
preferably only expressed when the polypeptide RNA is turned
on.
[0085] The vectors herein that contain the anti-termination nucleic
acid also contain a RNA recognition site. This would include the
Box B sequence in NutR or NutL for phage .lamda.N protein. This may
also include a Box A site if the anti-termination factor is a phage
protein. The RNA recognition site is engineered on the polypeptide
mRNA at the 5' end. The anti-termination nucleic acid then binds to
its RNA recognition site on the polypeptide mRNA and, in
conjunction with RNA polymerase and several host factors such as
nus factors, in the case of the N gene, forms an anti-termination
complex.
[0086] Preferably the Nut site is a NutR site, more preferably a
.lamda. NutR site, more preferably a complete BoxA and BoxB NutR or
a partial BoxB. The complete sequences of Nut sites, which include
Box A and Box B domains from phages .lamda., .phi.21, and P22, have
been published (Lazinski, Cell, 59: 207-218 (1989)). Box B is
responsible for binding an anti-terminator protein. Box A sequences
exist not only in phages, but in a variety of other
anti-termination operons, including the ribosomal RNA operons of E.
coli (Friedman and Olson, Cell, 34: 143-149 (1983); Li et al.,
Cell, 38: 851-860 (1984)). A conserved sequence of 8-12 nucleotides
proximal to the promoter in a natural operon, Box A is responsible
for binding a host elongation factor that interacts with the
anti-termination protein to stimulate anti-termination activity
(Greenblatt et al., Nature, 364: 401-406 (1993)).
[0087] The Box A domain should preferably match the anti-terminator
protein encoded by the gene used. Thus, if the anti-terminator
protein is the phage .lamda.N protein, one may choose a .lamda.,
NutL, or NutR Box A sequence, which differ slightly in nucleotide
sequence. Analogously, if the anti-termination protein is a phage
P22 N protein, one may choose a P22 Nut Box A sequence.
[0088] The source of the .lamda.N protein is one that is suitable,
including from the coding region of the N protein gene of pHE6
(Franklin and Bennett, Gene, 8: 107-119 (1979); EP 467,676
published Jan. 22, 1992) that is removed at the 7 HinfI restriction
site. Alternatively, one can use the commercially available plasmid
from Pharmacia LKB or a plasmid disclosed in EP 700,997, for
example.
[0089] In general, plasmid vectors containing replicon and control
sequences that are derived from species compatible with the host
cell are used in connection with prokaryotic hosts. The vector
ordinarily carries a replication site, as well as marking sequences
that are capable of providing phenotypic selection in transformed
cells. For example, E. coli is typically transformed using pBR322,
a plasmid derived from an E. coli species (see, e.g., Bolivar et
al., Gene, 2: 95 (1977)). pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR322 plasmid, or other
microbial plasmid or phage, also generally contains, or is modified
to contain, promoters that can be used by the microbial organism
for expression of the selectable marker genes.
[0090] The DNA encoding the polypeptide of interest herein may be
expressed not only directly, but also as a fusion with another
polypeptide, preferably a signal (leader) sequence or other
polypeptide having a specific cleavage site at the N-terminus of
the mature polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the polypeptide DNA
that is inserted into the vector. The heterologous signal sequence
selected should be one that is recognized and processed (i.e.,
cleaved by a signal peptidase) by the host cell. For, e.g.,
prokaryotic host cells that do not recognize and process the native
polypeptide signal sequence, the signal sequence is substituted by
a prokaryotic signal sequence selected, for example, from the group
consisting of the alkaline phosphatase, penicillinase, 1pp, or
heat-stable enterotoxin II leader sequences.
[0091] The vector also contains a transcription termination site.
The choice of termination site is usually not critical. Termination
sites are RNA sequences of about 50-100 bases downstream from the
translational stop site of a protein-coding sequence. Frequently,
RNA termination sites can fold to a hairpin structure. Termination
sites are recognized by RNA polymerase as a signal to cease
transcription (von Hippel, Science, 255: 809 (1992)). In eukaryotic
cells, the selection of termination site depends on the promoter to
which the genes are linked. However, in prokaryotic cells, RNA
polymerase recognizes virtually any prokaryotic termination site,
so the choice of termination site is not critical. In some vectors,
multiple termination sites are included in tandem.
[0092] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of prokaryotes. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative, bacteria.
[0093] Expression and cloning vectors also generally contain a
selection gene, also termed a selectable marker. This gene encodes
a protein necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not
survive in the culture medium. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. One example of a selection scheme
utilizes a drug to arrest growth of a host cell. Those cells that
are successfully transformed with a heterologous gene produce a
protein conferring drug resistance and thus survive the selection
regimen.
[0094] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
re-ligated in the form desired to generate the plasmids
required.
[0095] For analysis to confirm correct sequences in plasmids
constructed, the ligation mixtures are used to transform E. coli
K12 strain 294 (ATCC 31,446) or other strains, and successful
transformants are selected by ampicillin or tetracycline resistance
where appropriate. Plasmids from the transformants are prepared,
analyzed by restriction endonuclease digestion, and/or sequenced by
the method of Sanger et al., Proc. Natl. Acad. Sci. USA, 74:
5463-5467 (1977), Messing et al., Nucleic Acids Res., 9: 309
(1981), or Maxam et al., Methods in Enzymology, 65: 499 (1980).
[0096] Suitable prokaryotic cells useful as host cells herein
include bacteria, for example, archaebacteria and eubacteria,
especially eubacteria, and most preferably Enterobacteriaceae.
Examples of useful bacteria include Escherichia, Enterobacter,
Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus,
Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and
Paracoccus. These host cells may be lysogenic. Suitable E. coli
hosts include E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC
31,446), E. coli B, E. coli X1776 (ATCC 31,537), and E. coli JM105
(New England Biolabs). These examples are illustrative rather than
limiting. Mutant cells of any of the above-mentioned prokaryotic
cells may also be employed. It is, of course, necessary to select
the appropriate prokaryotic cells taking into consideration
replicability of the replicon in the cells of a prokaryote. For
example, E. coli, Serratia, or Salmonella species can be suitably
used as the host when well-known plasmids such as pBR322, pBR325,
pACYC177, or pKN410 are used to supply the replicon.
[0097] E. coli strain W3110 is a preferred host because it is a
common host strain for recombinant DNA product fermentations.
Preferably, the host cell should secrete minimal amounts of
proteolytic enzymes. For example, strain W3110 may be modified to
effect a genetic mutation in the genes encoding proteins, with
examples of such hosts including E. coli W3110 strain 1A2, which
has the complete genotype tonA.DELTA. (also known as .DELTA.fhuA);
E. coli W3110 strain 9E4, which has the complete genotype
tonA.DELTA. ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which
has the complete genotype tonA.DELTA. ptr3 phoA.DELTA.E15
.DELTA.(argF-lac)169 ompT.DELTA. degP41kan.sup.r; E. coli W3110
strain 37D6, which has the complete genotype tonA.DELTA. ptr3
phoA.DELTA.E15 .DELTA.(argF-lac)169 ompT.DELTA. degP41kan.sup.r
rbs7.DELTA. ilvG; E. coli W3110 strain 40B4, which is strain 37D6
with a non-kanamycin resistant degP deletion mutation; E. coli
W3110 strain 33D3, which has the complete genotype tonA ptr3 lacIq
LacL8 ompT degP kan.sup.r; E. coli W3110 strain 36F8, which has the
complete genotype tonA phoA .DELTA.(argF-lac) ptr3 degP kan.sup.R
ilvG+, and is temperature resistant at 37.degree. C.; an E. coli
strain having the mutant periplasmic protease(s) disclosed in U.S.
Pat. No. 4,946,783 issued Aug. 7, 1990; E. coli W3110 strain 52A7,
which has the complete genotype tonA.DELTA. (fhuA.DELTA.)
lon.DELTA. galE rpoHts (htpRts) .DELTA.clpP lacIq; E. coli W3110
strain 54C2, which has the complete genotype fhuA (tonA) lon galE
rpoHts (htpRts) clpP lacIq; and E. coli W3110 strain 59B9, which
has the complete genotype fhuA.DELTA. (tonA.DELTA.) lon.DELTA. galE
rpoHts (htpRts) .DELTA.clpP lacIq .DELTA.ompT .DELTA.(nmpc-fepE)
.DELTA.lacY.
[0098] Transformation means introducing DNA into an organism so
that the DNA is replicable, either as an extrachromosomal element
or by chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as
described in section 1.82 of Sambrook et al., Molecular Cloning: A
Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
1989), is generally used for prokaryotic cells that contain
substantial cell-wall barriers. Another method for transformation
employs polyethylene glycol/DMSO, as described in Chung and Miller,
Nucleic Acids Res., 16: 3580 (1988). Yet another method is the use
of the technique termed electroporation.
[0099] Host cells are transformed with the above-described
expression vectors of this invention and cultured in conventional
nutrient media modified as appropriate if promoters are induced.
Suitable media for this purpose are described generally, e.g., in
Sambrook et al., supra. Any other necessary supplements may also be
included at appropriate concentrations introduced alone or as
mixture with another supplement or medium such as a complex
nitrogen source. The pH of the medium may be any pH from about 5-9,
depending mainly on the host organism.
[0100] Gene expression may be measured in a sample by any means,
including indirectly or directly, for example, by conventional
northern blotting to quantitate the transcription of mRNA (Thomas,
Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)). Various labels
may be employed, most commonly radioisotopes, particularly
.sup.32P. However, other techniques may also be employed, such as
using biotin-modified nucleotides for introduction into a
polynucleotide. The biotin then serves as the site for binding to
avidin or antibodies, which may be labeled with a wide variety of
labels, such as radionuclides, fluorescers, enzymes, or the
like.
[0101] Procedures for observing whether an expressed or
over-expressed gene product is secreted are readily available to
the skilled practitioner. Once the culture medium is separated from
the host cells, for example, by centrifugation or filtration, the
gene product can then be detected in the cell-free culture medium
or cell culture by taking advantage of known properties
characteristic of the gene product. Such properties can include the
distinct immunological, enzymatic, or physical properties of the
gene product.
[0102] For example, if an over-expressed gene product has a unique
enzyme activity, an assay for that activity can be performed on the
culture medium used by the host cells or extracted cell pellets.
Moreover, when antibodies reactive against a given gene product are
available, such antibodies can be used to detect the gene product
in any known immunological assay (e.g., as in Harlowe et al.,
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press: New York, 1988)).
[0103] If the gene product is secreted, it can also be detected
using tests that distinguish polypeptides on the basis of
characteristic physical properties such as molecular weight. To
detect the physical properties of the gene product, all
polypeptides newly synthesized by the host cell can be labeled,
e.g., with a radioisotope. Common radioisotopes that can be used to
label polypeptides synthesized within a host cell include tritium
(.sup.3H), carbon-14 (.sup.14C), sulfur-35 (.sup.35S), and the
like. For example, the host cell can be grown in
.sup.35S-methionine or .sup.35S-cysteine medium, and a significant
amount of the .sup.35S label will be preferentially incorporated
into any newly synthesized polypeptide, including the
over-expressed heterologous polypeptide. The .sup.35S-containing
culture medium is then removed and the cells are washed and placed
in fresh non-radioactive culture medium. After the cells are
maintained in the fresh medium for a time and under conditions
sufficient to allow secretion of the .sup.35S-radiolabeled,
expressed heterologous polypeptide, the culture medium is collected
and separated from the host cells. The molecular weight of the
secreted, labeled polypeptide in the culture medium or
cell-associated in the cell pellet can then be determined by known
procedures, e.g., polyacrylamide gel electrophoresis. Such
procedures, and/or other procedures for detecting secreted gene
products, are provided, for example, in Goeddel, D. V. (ed.) 1990,
Gene Expression Technology, Methods in Enzymology, Vol. 185
(Academic Press), and Sambrook et al., supra.
[0104] For secretion of an expressed or over-expressed gene
product, the host cell is cultured under conditions sufficient for
secretion of the gene product. Such conditions include, e.g.,
temperature, nutrient, and cell density conditions that permit
secretion by the cell. Moreover, such conditions are those under
which the cell can perform basic cellular functions of
transcription, translation, and passage of proteins from one
cellular compartment to another, as are known to those skilled in
the art.
[0105] If the secretory elements are in place, the polypeptide of
interest is recovered from the periplasm or culture medium as a
secreted polypeptide. It is often preferred to purify the
polypeptide of interest from recombinant cell proteins or
polypeptides and from the anti-termination factor or GreA or GreB
protein to obtain preparations that are substantially homogeneous
as to the polypeptide of interest. As a first step, the culture
medium or lysate may be centrifuged to remove particulate cell
debris. The membrane and soluble protein fractions may then be
separated if necessary. The polypeptide may then be purified from
the soluble protein fraction and from the membrane fraction of the
culture lysate, depending on whether the polypeptide is
membrane-bound, is soluble, or is present in an aggregated form.
The polypeptide thereafter is solubilized and refolded, if
necessary, and is purified from contaminant soluble proteins and
polypeptides.
[0106] One method for isolating exogenous polypeptides from a
complex biological mixture containing polypeptides and
non-polypeptides contained in a fermentation broth involves contact
of reagents with the cells, preferably the cell culture, containing
the polypeptide in a non-native conformation, so that an aqueous
extraction/isolation can take place. Preferably, the method entails
direct addition of reagents to the fermentation vessel after the
polypeptide has been produced recombinantly, thereby avoiding extra
steps of harvesting, homogenization, and centrifugation to obtain
the polypeptide. While the remaining particulates can be removed by
Gaulin homogenization and re-suspension, filtration, or a
combination thereof, this method utilizes a multiple-phase
extraction system for purifying recombinant polypeptides from the
remaining particulates.
[0107] The following procedures are exemplary of suitable
purification procedures: fractionation on immunoaffinity or
ion-exchange columns; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
and gel filtration using, for example, SEPHADEX.TM. G-75 medium
from Amersham Biosciences.
[0108] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All literature and patent
citations mentioned herein are expressly incorporated by
reference.
EXAMPLE 1
Effect of .lamda.N Anti-termination System on TPO Production
(Shake-Flask)
Materials and Methods:
Description and Construction of E. coli Expression Vectors
[0109] The plasmids pMP331, pMP843, pMP871, pMP931, pMP951, pMP982,
pMP945, pMP1016, pMP1086, pMP1099, pMP1201, and pMP1217 are all
designed to express full-length mature TPO (deSauvage et al.,
Nature, 369: 533-538 (1994)) in the E. coli cytoplasm using a
pBR322-based vector (Bolivar et al., Gene, 2: 95-113 (1977)). The
332-amino-acid TPO coding sequence is preceded in all plasmids by a
leader consisting of the first seven amino acids of the heat-stable
enterotoxin II signal sequence (Picken et al., Infect. Immun., 42:
269-275(1983)), followed by eight histidine residues, and finally
the thrombin cleavage site IEPR (SEQ ID NO:6). Transcription in the
plasmids pMP331, pMP843, pMP871, pMP931, pMP945, pMP951, pMP982,
and pMP1217 is under the control of the trp promoter (Yanofsky et
al., Nucleic Acids Res., 9: 6647-6668 (1981)), while pMP1016,
pMP1086, pMP1099, and pMP1201 use the AP promoter (Kikuchi et al.,
Nucleic Acids Res., 9: 5671-5678 (1981)). Just downstream of the
TPO coding sequence is situated the .lamda.t.sub.o transcriptional
terminator (Scholtissek et al., Nucleic Acids Res., 15: 3185
(1987)).
[0110] Additional genetic elements on pMP843, pMP871, pMP931, and
pMP945 include the partial .lamda. nutR site Box B (or B box),
while plasmids pMP951, pMP982, pMP1086, pMP1201, and pMP1217 have
the complete A nutR site of Boxes A and B (Olson et al., Cell, 31:
61-70 (1982)) at the site where the TPO-encoding message begins.
Plasmids pMP871, pMP931, pMP945, pMP951, pMP982, pMP1086, pMP1201,
and pMP1217 also contain the gene for .lamda.N protein (Franklin,
J. Mol. Biol., 181: 75-84 (1984)). Finally, plasmids pMP982,
pMP1086, pMP1099, and pMP1201 contain the sequence for a rare
arginine tRNA, the argU gene (dnaY gene) (Garcia et al., Cell, 45:
453-459 (1986)).
[0111] The plasmid pDR1 is designed to express the protein GreB
(Borukhov et al., Cell, supra) under the control of the tacII
promoter (DeBoer et al., supra). The backbone of this plasmid is
pACYC177 (Chang et al., J. Bacteriol., 134: 1141-1156 (1978); Rose,
Nucleic Acids Res., 16: 356 (1988)), which allows it to be
compatibly maintained in the same E. coli cell with the
pBR322-based plasmids.
Plasmid pMP331
[0112] The plasmid pMP331 is a derivative of the TPO expression
plasmid pMP202 (WO 95/18858 published Jul. 13, 1995). Briefly,
pMP202 is designed to express the first 155 amino acids of TPO
downstream of a leader comprising seven amino acids of the STII
signal sequence, eight histidines, and a thrombin cleavage site.
The plasmid pMP331 extends the TPO coding sequence from 155 to 332
amino acids.
[0113] Three DNA fragments were ligated together to make pMP331 as
shown in FIG. 1, the first of which was the large fragment of the
vector pdh108 previously cut with XbaI and StuI. pdh108 is derived
from the vector pHGH207-1 (DeBoer et al., Promoters: Structure and
Function (Praeger: New York, 1982), pp. 462-481) and contains the
.lamda.t.sub.o transcriptional terminator downstream of the trp
promoter. The second part was the 431-base-pair XbaI-BamHI fragment
from the plasmid pMP202 encoding the first 122 amino acids of TPO.
The third part was an approximately 669-base-pair BamHI-RsaI
fragment from phmpll (deSauvage et al., Nature, 369: 533-538
(1994)) encoding the last half of the TPO gene.
Plasmid pMP843
[0114] The plasmid pMP843 is the result of adding the .lamda. nutB
box (nutR Box B) downstream of the trp promoter in plasmid pMP331.
Two fragments were ligated together to produce pMP843 as shown in
FIG. 2, the first of which was pMP331 in which the small SpeI-SacI
fragment had been removed. The second was a 642-base-pair SpeI-SacI
fragment obtained by ligating a synthetic DNA duplex to the
582-base-pair XbaI-SacI fragment from pMP331. The synthetic DNA
duplex had the following sequence: TABLE-US-00001
5'-CTAGTTAACTAGTACGCATTCCAGCCCTGAAAAAGGGCAAAGTTCAC GTAAAAAGGATAT
AATTGATCATGCGTAAGGTCGGGACTTTTTCCCGTTTCAAGTGCATTTTT CCTATAGATC-5'
(SEQ ID NOS:7 and 8, respectively)
Plasmid pMP871
[0115] The plasmid pMP871 is derived from pMP843 and contains the
gene for .lamda.N protein polycistronically coupled downstream of
the TPO coding sequence. pMP871 was constructed as shown in FIG. 3,
by ligating together three DNA fragments, the first of which was
the vector pMP843 from which the small SacI-NheI fragment had been
removed. The second is an approximately 500-base-pair SacI-FokI
fragment prepared by pre-ligating a synthetic DNA duplex to the
459-base-pair SacI-AlwNI fragment from pMP843 encoding amino acids
174-327 of TPO. The synthetic DNA duplex had the following
sequence: TABLE-US-00002
5'-CTGTCTCAGGAAGGGTAAGCTTTTATGGATGCACAAACAC
TTAGACAGAGTCCTTCCCATTCGAAAATACCTACGTGTTTGTGCGGC-5' (SEQ ID NOS:9
and 10, respectively)
[0116] The final part in the ligation was an approximately
657-base-pair FokI-NheI fragment from the commercial vector
pPL-.lamda. (Pharmacia Biotech Inc.) encoding amino acids 7-107 of
.lamda.N protein.
Plasmid pMP931
[0117] The plasmid pMP931 is a derivative of pMP843 in which the
.lamda.N gene is placed under the control of the tacII promoter. As
shown in FIG. 4, pMP931 was constructed by ligating together three
DNA fragments. The first of these was the vector pMP843 in which
the small ClaI-NheI fragment had been removed. The second was an
approximately 100-base-pair HinPI-HindIII fragment from the plasmid
pKMTacII (DeBoer et al., (1983), supra) containing the tacII
promoter. The third part in the ligation was a 684-base-pair
HindIII-NheI fragment from pMP871 encoding the .lamda.N gene.
Plasmid pMP945
[0118] The plasmid pMP945 is a derivative of pMP931 in which
.lamda.N gene expression from the tacII promoter is accompanied by
a full Shine-Dalgarno sequence for higher translation levels. As
shown in FIG. 5, this plasmid was constructed by ligating together
three DNA fragments, the first of which was the large vector
fragment obtained by digesting pMP931 with HindIII and NheI. The
second fragment was a synthetic DNA duplex with the following
sequence: TABLE-US-00003 5'-AGCTTAGGATTCTAGAATTATGGATGCACAAACAC
ATCCTAAGATCTTAATACCTACGTGTTTGTGCGGC- 5' (SEQ ID NOS:11 and 12,
respectively)
The last part was the 657-base-pair FokI-NheI fragment used in the
construction of pMP871. Plasmid pMP951
[0119] The plasmid pMP951 is derived from pMP931 and additionally
contains the full nut site (both Boxes A and B). As shown in FIG.
6, pMP951 was constructed by ligating together two DNA fragments.
The first of these was the vector pMP931 from which the small
AatII-XbaI fragment had been removed. The second was prepared by
pre-ligating a synthetic DNA duplex to the 322-base-pair AatII-SpeI
fragment from pMP931. The synthetic DNA duplex had the following
sequence: TABLE-US-00004 5'-CTAGTTAACTAGTACGCAACGCTCTTACACAT- (SEQ
ID NO:13) TCCAGCCCTGAAAAAGGGCAAAGTTCACGTAAAAAG GATAT
AATTGATCATGCGTTGCGAGAATGTGTAAGGTCGG- (SEQ ID NO:14)
GACTTTTTCCCGTTTCAAGTGCATTTTTCCTATAGA TC-5'
Plasmid pMP982
[0120] The plasmid pMP982 is a derivative of pMP951 with the
addition of the argU gene on the plasmid downstream of the .lamda.N
gene. As shown in FIG. 7, this plasmid was constructed by ligating
together two DNA fragments. The first of these was the vector
pMP951 in which the small NheI-SphI fragment had been removed, and
in which the NheI site had been blunted by treatment with DNA
polymerase I (Klenow). The second part was a 440-base-pair
ClaI-SphI fragment from pST141 in which the ClaI site had been
blunted by treatment with DNA polymerase I (Klenow). pST141 is a
derivative of the plasmid pHGH207-1 (DeBoer et al., (1983), supra),
and this fragment only encodes the argU gene (Garcia et al., Cell,
45: 453-459 (1986)).
Plasmid pMP1016
[0121] The plasmid pMP1016 is a derivative of pMP331 in which the
trp promoter has been replaced with the AP promoter. This plasmid
was constructed as shown in FIG. 8 by ligating together two DNA
fragments. The first of these was the vector pST182 in which the
small XbaI-SphI fragment had been removed. The plasmid pST182 is a
derivative of phGH1 (Chang et al., Gene, 55: 189-196 (1987)), and
this latter vector could be used instead to generate this DNA
fragment. The second part in the ligation was a 1791-base-pair
XbaI-SphI fragment from pMP331 encoding the TPO gene and the
.lamda.t.sub.o transcriptional terminator.
Plasmid pMP1086
[0122] The plasmid pMP1086 is derived from pMP982 and results in
the AP promoter being substituted for the trp promoter. Three DNA
fragments were ligated together to construct pMP1086,as shown in
FIG. 9. The first of these was the vector pST182 from which the
small SpeI-SphI fragment had been removed. The plasmid pST182 is a
derivative of phGH1 (Chang et al., Gene, supra) and this latter
vector could be used instead to generate this fragment. The second
part in the ligation was a 647-base-pair SpeI-SacI fragment from
pMP982 encoding the nut site and the first 173 amino acids of TPO.
The third part was a 1809-base-pair SacI-SphI fragment from pMP982
encoding TPO amino acids 174-332, and containing the .lamda.t.sub.o
terminator, .lamda.N gene, and argU gene.
Plasmid pMP1099
[0123] The plasmid pMP1099 is derived from pMP1016 and additionally
contains the argU gene downstream of the .lamda.t.sub.o
transcriptional terminator. As shown in FIG. 10, it was constructed
by ligating together two DNA fragments. The first of these was the
vector pMP1016 in which the small SacI-SphI fragment had been
removed. The second was a 1020-base-pair SacI-SphI fragment from
the plasmid pMP591 encoding the last 159 amino acids of TPO, the
.lamda.t.sub.o transcriptional terminator, and the argU gene. The
plasmid pMP591 is a derivative of pMP331 with the addition of the
argU gene just downstream of the .lamda.t.sub.o transcriptional
terminator.
Plasmid pMP1201
[0124] The plasmid pMP1201 is a derivative of pMP1086 in which
.lamda.N gene expression from the tacII promoter is accompanied by
a full Shine-Dalgarno sequence for higher translation levels. As
shown in FIG. 11, this plasmid was constructed by ligating together
three DNA fragments, the first of which was the large vector
fragment obtained by digesting pMP1086 with SacI and SphI. The
second was a 1007-base-pair SacI-ClaI fragment from pMP945
containing the tacII promoter with full Shine-Dalgarno and most of
the .lamda.N gene. The final piece was a 814-base-pair ClaI-SphI
fragment obtained by digesting pMP1086 completely with SphI, and
partially with ClaI.
Plasmid pMP1217
[0125] The plasmid pMP1217 is a derivative of pMP951 in which
.lamda.N gene expression from the tacII promoter is accompanied by
a full Shine-Dalgarno sequence for higher translation levels. As
shown in FIG. 12, this plasmid was constructed by ligating together
two DNA fragments. The first of these was the large vector fragment
of pMP951 after digestion with HindIII and NheI. The second was a
696-base-pair HindIII-NheI fragment from pMP945 encoding a full
Shine-Dalgarno sequence preceding the .lamda.N gene.
Plasmid pJJ142
[0126] The plasmid pJJ142 is an intermediate in the construction of
pDR1 and was prepared by ligating together two DNA fragments as
shown in FIG. 13. The first of these was the plasmid pACYC177 in
which the small AatII-HincII fragment had been removed. The second
part in the ligation was the 1021-base-pair AatII-ClaI fragment
from pJJ41 (U.S. Pat. No. 5,639,635) in which the ClaI site had
been blunted by treatment with DNA polymerase I (Klenow). This
latter fragment encodes the tacII promoter followed by the dsbC
gene.
Plasmid pDR1
[0127] The plasmid pDR1 is designed to express GreB under the
control of the tacII promoter. pDR1 was constructed as shown in
FIG. 14 by the ligation of two DNA fragments. The first of these
was the vector pJJ142 in which the small XbaI-PstI fragment had
been removed. The second part was a 488-base-pair XbaI-PstI
fragment containing the greB gene. This fragment was prepared by
first amplifying the greB coding sequence by PCR using E coli
chromosomal DNA and then digesting this mixture with XbaI and PstI.
The following primers were used for this step: TABLE-US-00005
5'-CCCCCCCCCTCTAGAAAAATGAAAACTCCTCTG (SEQ ID NO:15) GTAACGCGGGAAGGG
5'-CCCCCCCCCCTGCAGTTACGGTTTCACGTACTC (SEQ ID NO:16) GATAGC
Plasmid pDR3
[0128] The plasmid pDR3 is designed to express GreA under the
control of the tacII promoter. pDR3 was constructed as shown in
FIG. 15 by the ligation of two DNA fragments. The first of these
was the vector pJJ142 in which the small XbaI-PstI fragment had
been removed. The second part was a 491-base-pair XbaI-PstI
fragment containing the greA gene. This fragment was prepared by
first amplifying the greA coding sequence by PCR using E coli
chromosomal DNA and then digesting this mixture with XbaI and PstI.
The following primers were used for this step: TABLE-US-00006
5'-CCCCCCCCCTCTAGAATTCTATGCAAGCTATTC (SEQ ID NO:17) CGATGACCTTA
5'-CCCCCCCCCCTGCAGTTACAGGTATTCCACCTT (SEQ ID NO:18) AAT
E. coli Host
[0129] The strain 52A7, which is a derivative of W3110 (ATCC
27,325) having the genotype tonA.DELTA. (fhuA.DELTA.) lon.DELTA.
galE rpoHts (htpRts) AclpP lacIq, was used for the transformation
experiments.
Transformation and Culturing
[0130] For determining if .lamda.N anti-termination had any effects
on the truncation and 10Sa tagging of TPO, the trp expression
vector pMP331 and the anti-termination plasmids with low (pMP951)
and high levels (pMP1217) of N expression were transformed into
strain 52A7 using standard procedures. All these transformants were
grown in Luria Broth (LB) media with ampicillin overnight at
30.degree. C., and then diluted 20 fold into M9 with casamino acids
media also containing ampicillin. After approximately 6 hours at
30.degree. C. with shaking, the optical density of the cultures
(600 nm) was between 2 and 2.5, and isopropyl
.beta.-D-thiogalactopyranoside (IPTG) (1 mM) was added to the two
anti-termination cultures pMP951 and pMP1217. All cultures were
then grown an additional 15 hours with shaking at 30.degree. C.
Samples were then removed, prepared as described in Yansura et al.,
Methods in Mol. Biol., 62: 44-62 (1997), and analyzed by
SDS-PAGE.
[0131] TPO expression with the AP promoter was then tested to see
if similar results would be obtained with .lamda.N
anti-termination. The AP expression plasmid pMP1099 as well as the
anti-termination plasmids with low-(pMP1086) and high-level
(pMP1201) N expression were first transformed into the E. coli
strain 52A7. Transformants were first grown in LB media containing
ampicillin overnight at 30.degree. C., and then diluted 100 fold
into a phosphate-limiting media called C.R.A.P., which also
contains ampicillin. (C.R.A.P. medium contains 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g NaCitrate-2H.sub.2O, 1.07 g KCl,
5.36 g yeast extract (Certified), 5.36 g HY-CASE.RTM. SF refined
acid-hydrolyzed casein (Quest International), 110 mL 1M morpholino
propane sulfonic acid (MOPS) pH 7.3, 11 mL of a 50% glucose
solution, and 7 mL 1M MgSO.sub.4 in a final volume of 1 liter).
After growth at 30.degree. C. for approximately 6 hours, the
cultures reached an optical density (600 nm) of between 1.5 and
2.0, and IPTG (1 mM) was added to the two anti-termination
plasmids. Growth at 30.degree. C. was continued for another 15
hours, at which time samples were removed, prepared as described
for the trp vectors, and analyzed by SDS-PAGE.
Results:
Production of TPO using Control Plasmids pMP331 and pMP1099
[0132] TPO (de Sauvage et al., Nature, 369: 533-538 (1994)) was
expressed in the cytoplasm of E. coli strain 52A7 under the control
of both the trp (pMP331) and the AP (pMP1099) promoters with a
small amino-terminal polyhis leader to provide for easy
purification. Induction of either promoter trp or AP led to the
production of TPO-related protein that could be detected by
Coomassie-stained whole-cell extracts separated on an SDS
polyacrylamide gel (SDS-PAGE), as shown in FIG. 16A.
[0133] Besides the expected protein band for the full-length
polyhis leader-TPO at 37 kD, several other induced protein bands of
lower molecular mass were noted. The most noticeable of these had
masses of approximately 25, 18, and 14 kD. To determine if these
lower-molecular-weight bands were TPO-related, the whole cell
lysate was again separated by SDS-PAGE, transferred to
nitrocellulose, and probed with an agent that binds to the polyhis
motif on the leader (INDIA.TM. metal-chelated histidine probe bound
to HRP (HisProbe-HRP) offered by Pierce Chemical Company) as shown
in FIG. 16B. The results verified that the previously noted
lower-molecular-weight bands were indeed TPO-related, and also
revealed a much more severe truncation problem occurring on the
C-terminal end of the protein.
[0134] To ascertain if these multiple truncated forms of TPO
resulted from degradation of the polyhis leader containing
full-length protein or were initially synthesized in this way, a
pulse-chase analysis was performed using the same plasmid
construct. Without limitation to any one theory, the results
suggest that the latter case was the more likely explanation. The
formation of truncated forms of TPO can clearly be seen showing up
in the very early chase times of 0.5 and 1 minutes and remaining
throughout the experiment up to 6 minutes. There was no obvious
movement of TPO-related protein from full-length to truncated forms
or vice-versa, suggesting that the lower-molecular-weight forms
were produced directly during protein synthesis.
[0135] One possible explanation that needed to be ruled out was
plasmid instability, particularly deletions in the TPO-coding
sequence. Therefore, the plasmid DNA was isolated by induction
culturing, with the resulting DNA subjected to PAGE analysis after
digestion with several restriction endonucleases. In addition,
individual colonies from the induction culture were analyzed in a
similar fashion. In all cases there was no evidence of any plasmid
alterations, particularly in the TPO-coding sequence.
[0136] Finally, the possibility of producing truncated TPO
fragments by the translation of truncated mRNA was investigated.
Such protein fragments had previously been shown to contain a
C-terminal, 11-amino-acid tag encoded by a small open reading frame
in the 10Sa RNA (Tu et al., supra; Keiler et al., supra). A
polyclonal antibody directed against the chemically-synthesized tag
was used to probe whole cell extracts from TPO induction cultures.
The results in FIG. 16C clearly show that the majority of the
truncated TPO fragments contained the 10Sa 11-amino-acid tag, and
suggest, without limitation to any one theory, that the TPO mRNA is
truncated or damaged.
Production of TPO using Anti-Termination System and Effect on 10Sa
Tagging
[0137] Although the expression of full-length TPO with the
N-terminal polyhis leader was relatively high with both the trp and
the AP promoters, the accumulation of truncated forms of TPO
greatly interfered with protein purification. Since all of the
truncated forms have the N-terminal polyhis leader, they co-purify
with the full-length protein on a metal chelation column.
Elimination of these truncated forms therefore becomes an important
factor in the purification and production of a protein such as
TPO.
[0138] Despite the failure of rrnB anti-termination to work for
this purpose (Makrides, supra), a different transcription
anti-termination agent, .lamda.N, was used in this Example to
minimize or eliminate the truncation and 10Sa tagging of TPO by
promoting transcriptional readthrough of intragenic termination
signals within the TPO coding sequence.
Incorporating .lamda.N Anti-Termination
[0139] To incorporate .lamda.N anti-termination into the trp and AP
expression systems for TPO, the N utilization sequence (nutL) was
first inserted into the plasmids at locations corresponding to the
beginning of the TPO mRNA. The actual design of these sequences,
including the upstream promoters and the downstream polyhis leader,
is shown in FIG. 17. The .lamda.N gene was then inserted into these
plasmids downstream of the TPO coding sequence and the
.lamda.t.sub.o transcriptional terminator. N expression was placed
under the control of the tacII promoter (DeBoer et al., (1983),
supra), and plasmids with and without the tacII Shine-Dalgarno
sequences were constructed. This variation in the translation of N
provided an easy way to look at two different expression levels of
N, and their subsequent effects on TPO expression. Partial
sequences showing the tacII promoter with and without the
Shine-Dalgarno sequence are shown in FIG. 18.
Effects of .lamda.N Anti-Termination on TPO Expression
[0140] Coomassie staining of the SDS gels for the trp expression
plasmids pMP331, pMP951, and pMP1217 revealed primarily an increase
in the expression of full-length TPO with both low-level (pMP951)
and high-level (pMP1217) N expression as compared with the control
(pMP331) as shown in FIG. 19A. Analysis of the SDS gel, after
transferring to nitrocellulose and probing with HisProbe-HRP,
showed not only an increase in the expression of full-length TPO,
but also a significant decrease in the formation of truncated forms
of TPO for both anti-termination plasmids (FIG. 19B). Finally, an
analysis of the gel after transferring to nitrocellulose and
probing with antibody to the 10Sa tag also showed a significant
decrease in the accumulation of truncated and 10Sa-tagged forms of
TPO with both anti-termination plasmids (FIG. 19C).
[0141] Coomassie staining of the SDS gels for the AP expression
plasmids pMP1099, pMP1086, and pMP1201 showed an increase in the
level of full-length TPO with both anti-termination plasmids as
compared to the control plasmid pMP1099, as was seen with the trp
plasmids (FIG. 20A). After transferring to nitrocellulose and
probing with HisProbe-HRP, one can also see an increase in the
level of full-length TPO as well as a significant decrease in the
expression of truncated forms of TPO (FIG. 20B). In a similar
analysis, probing with the anti-10Sa antibody showed a decrease in
the accumulation of truncated and 10Sa-tagged forms of TPO with
both anti-termination plasmids (FIG. 20C).
CONCLUSION
[0142] It is clear from the above shake-flask results that the
presence of .lamda.N anti-termination with low or high amounts of N
proteins using either promoter increased the titer of the
polypeptide. Further, the presence of the .lamda.N protein
anti-termination system in low or high amounts with either promoter
reduced the amount of 10Sa-tagged protein made by the cells.
EXAMPLE 2
Effect of .lamda.N Anti-Termination System on TPO Production
(Fermentor)
Materials and Methods:
Transformation
[0143] The strain 52A7 was transformed with each of pMP331, pMP951,
pMP1099, or pMP1086, each of which is described above in Example 1,
using standard procedures involving ampicillin or tetracycline as
appropriate.
Culture of Transformed Cells
[0144] Example for pMP331 and pMP951:
[0145] A 10-liter fermentation was carried out in the following
medium, with modifications as noted. A bag of salts suitable for a
10-liter fermentation contained the following salts: TABLE-US-00007
Salt Grams Ammonium Sulfate 50.0 Potassium Phosphate, dibasic 60.0
Sodium Phosphate, monobasic, dihydrate 30.0 Sodium Citrate,
dihydrate 10.0
In addition to the salts, 5 g L-isoleucine and 3 mL of a 25%
solution of PLURONIC.RTM. L-61 antifoam polyol (BASF Corporation)
were added to the fermentor. The fermentor was sterilized with
these components and 5-6.5 liters of deionized water. After the
fermentor and contents cooled down, the post-sterile ingredients
were added. The post-sterile ingredients consisted of 15 mL of a
50% glucose solution, 70 mL of 1 M magnesium sulfate, 5 mL of trace
metals (recipe below), 250 mL of 20% HY-CASE.RTM. acid-hydrolyzed
casein solution, 250 mL of 20% yeast extract solution, and 250 mL
of a 2 mg/mL ampicillin solution. The starting volume in the
fermentor, after inoculation, was usually 8.5 liters.
[0146] The fermentor was inoculated with 500 mL of a 16-20-hour LB
culture that had been grown with agitation at 30.degree. C. The LB
culture was grown in the presence of ampicillin. The 10-liter
culture was agitated at 750 rpm and aerated at 10 slpm. The culture
pH was maintained at 7.0-7.3 by the automatic addition of ammonium
hydroxide, and the temperature was maintained at 30.degree. C. When
the initial glucose in the culture was exhausted, a glucose feed
was started and maintained at such a rate as to prevent starvation
and also to avoid accumulation of glucose in the medium.
[0147] Culture growth was monitored by measuring the optical
density (O.D.) at a wavelength of 550 nm. When the culture O.D.
reached 25-35, 25 mL of a 25 mg/mL solution of 3-.beta.-indole
acrylic acid (IAA) and 2-50 mL of a 200 mM IPTG solution (for
pMP951 only) were added. The cell paste was harvested via
centrifugation 14-18 hours after IAA addition. TABLE-US-00008 Trace
Element Amounts Hydrochloric Acid 100 mL Ferric Chloride
hexahydrate 27 g/L Zinc Sulphate heptahydrate 8 g/L Cobalt Chloride
hexahydrate 7 g/L Sodium Molybdate 7 g/L Cupric Sulphate
pentahydrate 8 g/L Boric Acid 2 g/L Manganese Sulphate monohydrate
5 g/L Deionized Water to 1 liter
[0148] Example for pMP1099 and pMP1086:
[0149] A 10-liter fermentation was carried out in the following
medium, with modifications as noted. A bag of salts suitable for a
10-liter fermentation contained the following salts: TABLE-US-00009
Salt Grams Ammonium Sulfate 50.0 Potassium Phosphate, dibasic 26.0
Sodium Phosphate, monobasic, dihydrate 13.0 Sodium Citrate,
dihydrate 10.0 Potassium Chloride 15.0
In addition to the salts, 5 g L-isoleucine and 3 mL of a 25%
solution of PLURONIC.RTM. L-61 antifoam polyol (BASF Corporation)
were added to the fermentor. The fermentor was sterilized with
these components and 5-6.5 liters of deionized water. After the
fermentor and contents cooled down, the post-sterile ingredients
were added. The post-sterile ingredients consisted of 15 mL of a
50% glucose solution, 70 mL of 1 M magnesium sulfate, 5 mL of trace
metals (recipe below), 250 mL of 20% HY-CASE.RTM. acid-hydrolyzed
casein solution, 250 mL of 20% yeast extract solution, and 250 mL
of a 2 mg/mL-ampicillin solution. The starting volume in the
fermentor, after inoculation, was usually 8.5 liters.
[0150] The fermentor was inoculated with 500 mL of a 16-20-hour LB
culture that had been grown with agitation at 30.degree. C. The LB
culture was grown in the presence of ampicillin. The 10-liter
culture was agitated at 750 rpm and aerated at 10 slpm. The culture
pH was maintained at 7.0-7.3 by the automatic addition of ammonium
hydroxide, and the temperature was maintained at 30.degree. C. When
the initial glucose in the culture was exhausted, a glucose feed
was started and maintained at such a rate as to prevent starvation
and also to avoid accumulation of glucose in the medium.
[0151] Culture growth was monitored by measuring the O.D. at a
wavelength of 550 nm. When the culture O.D. reached 25-35, 2-50 mL
of a 200 mM IPTG solution (for pMP1086 only) was added. The cell
paste was harvested via centrifugation 20-30 hours after
inoculation. TABLE-US-00010 Trace Element Amounts Hydrochloric Acid
100 mL Ferric Chloride hexahydrate 27 g/L Zinc Sulphate
heptahydrate 8 g/L Cobalt Chloride hexahydrate 7 g/L Sodium
Molybdate 7 g/L Cupric Sulphate pentahydrate 8 g/L Boric Acid 2 g/L
Manganese Sulphate monohydrate 5 g/L Deionized Water to 1 liter
Generation of Polyclonal and Monoclonal Antibodies to the 10Sa
Peptide:
[0152] The following peptide was synthesized for generating
antibodies: CAANDENYALAA (SEQ ID NO:19). The N-terminal cysteine is
present to allow conjugation of the peptide to either KLH or
soybean trypsin inhibitor, or another suitable conjugation partner.
The remaining residues are encoded by the ssrA gene from E. coli
and translate to give the 10Sa peptide. The above peptide was
synthesized and conjugated to KLH by Zymed Corporation and used as
the antigen to raise antibodies in both rabbits and mice.
[0153] Specific antibodies to the 10Sa peptide were obtained by
taking either serum from rabbits injected with the antigen or
ascites fluid from mice. The serum or ascites fluid was passed over
an affinity column that had the synthetic 10Sa peptide bound to it.
The specific antibodies were eluted from the 10Sa affinity column
using low pH.
Quantitation of 10Sa-Tagged and Full-Length TPO:
[0154] TPO fusion proteins made from the plasmids pMP331, pMP951,
pMP1099, and pMP1086 were extracted from whole cell pellets using a
buffer containing 7.5 M guanidine HCl, 0.1 M sodium sulfite, 0.02 M
sodium tetrathionate, and 50 mM Tris buffer, pH 8.0. Extractions
were allowed to continue 1-16 hours with stirring at room
temperature. The extracted solution was then clarified with
centrifugation and the supernatant dialyzed 1-16 hours at 4.degree.
C. against a buffer containing 6 M GuHCl and 20 mM Tris buffer, pH
7.5. The polyhis-containing proteins from the dialysate were
purified via a chelating column (Talon Metal Affinity Resins,
Clontech). The protein eluted from the column was run on SDS-PAGE,
transferred to nitrocellulose, and probed with a polyclonal
antibody raised against either TPO.sub.153 (WO 95/18858 published
Jul. 13, 1995) or the 10Sa peptide. The blots were then scanned
using an optically enhanced laser densitometer (PDI Inc., model
325oe). The peak areas for the full-length TPO, as well as the
other TPO species that cross-react with the TPO.sub.153 polyclonal
antibody, were determined.
Results:
[0155] The results from these analyses are shown in Table 1. The
ratio of full-length TPO to all of the TPO species was calculated
and is reported as % TPO. In addition, the total peak area detected
on the blot probed with the polyclonal antibody raised to the 10Sa
peptide was also calculated and is reported as 10Sa tag.
TABLE-US-00011 TABLE 1 Effect of Plasmid Construct on TPO332
Accumulation and 10Sa Tagging Plasmid Nut Site N Protein % TPO 10Sa
Tag pMP331 - - 4.5 11.5 pMP951 + + 49.0 2.6 pMP1099 - - 50.0 4.6
pMP1086 + + 73.0 1.6
[0156] The data in Table 1 show that expressing TPO from a plasmid
with the .lamda.N anti-termination system resulted in an increased
percentage of TPO that is full-length TPO and a decrease in
accumulated 10Sa tagged TPO. This is seen whether the AP or the trp
promoter is used to control TPO expression.
EXAMPLE 3
Effect of GreA or GreB on TPO Production (Shake-Flask)
Materials and Methods:
Transformations
[0157] The strain 59B9 (W3110 fhuA.DELTA. (tonA.DELTA.) lon.DELTA.
galE rpoHts (htpRts) .DELTA.clpP lacIq .DELTA.ompT .DELTA.
(nmpc-fepE) .DELTA.lacY) was transformed with each of pMP331,
pMP951, pMP1217, pMP1099, pMP1086, or pMP1201 either alone or in
combination with pDR1 or pDR3, each of which is described above in
Example 1, using standard procedures.
Culture of Transformed Cells
[0158] The transformed cells were grown in LB media with ampicillin
and kanamycin (when co-transformed with pDR1 or pDR3 only) at
30.degree. C. with shaking overnight and then diluted 50-fold into
shake-flask culture medium containing ampicillin and grown at
30.degree. C. with shaking. Transformants containing the plasmids
pMP331, pMP951, or pMP1217 were grown in THCD medium with the
appropriate antibiotic until they reached an O.D.550 of 1-2, at
which time IAA (50 .mu.g/ml final concentration) and IPTG (1 mM,
final concentration) (for pMP951 and pMP1217 only) were added to
the culture. All cultures were grown for a total of 24 hours.
Samples were then removed and prepared for SDS-PAGE.
[0159] THCD medium contains 1.86 g Na.sub.2HPO.sub.4, 0.93 g
NaH.sub.2PO.sub.4--H.sub.2O, 3.57 g (NH.sub.4).sub.2SO.sub.4, 0.71
g NaCitrate-2H.sub.2O, 1.07 g KCl, 5.36 g yeast extract (Difco.RTM.
Bacto.RTM. brand, #0127-01-7), 5.36 g casamino acids (Difco.RTM.
Bacto.RTM. brand, #0230-17-3), 7 mL of 1M MgSO.sub.4, 11 mL of a
50% glucose solution, and 110 mL 1 M MOPS, pH 7.3, in a final
volume of 1 liter.
[0160] Transformants containing the plasmids pMP1099, pMP1086, or
pMP1201 with or without either pDR1 or pDR3 were grown in C.R.A.P.
medium with the appropriate antibiotics until they reached an
O.D.550 of 1-2, at which time IPTG (1 mM final concentration) was
added to all of the cultures except for the one containing pMP1099
alone. All cultures were grown for a total of 24 hours. Samples
were then removed and prepared for SDS-PAGE.
Quantitation of 10Sa-Tagged and Full-Length TPO
[0161] Samples from the shake flask cultures described above were
prepared and run on SDS-PAGE, transferred to nitrocellulose, and
probed with a polyclonal antibody raised against either TPO.sub.153
or the 10Sa peptide. The blots were then scanned using an optically
enhanced laser densitometer (PDI, Inc., model 325oe). The peak
areas for the full-length TPO, as well as the other TPO species
that cross-react with the TPO.sub.153 polyclonal antibody, were
determined.
[0162] The same set of shake flask samples was also run on SDS-PAGE
and stained with Coomassie Blue. The gels were scanned using an
optically enhanced laser densitometer (PDI, Inc., model 325oe) and
the percentage of total cell protein represented as full-length TPO
was calculated.
Results:
[0163] The results from these analyses are shown in Table 2, where
SD signifies the Shine-Dalgarno sequence. The ratio of full-length
TPO to all of the TPO species was calculated and is reported as %
TPO. In addition, the total peak area detected on the blot probed
with the polyclonal antibody raised to the 10Sa peptide was also
calculated and is reported as 10Sa tag. The percentage of total
cell protein represented as full-length TPO is shown as % of total
protein. TABLE-US-00012 TABLE 2 Effect of Plasmid Construct on
TPO332 Accumulation and 10Sa Tagging Plasmid(s) Nut Site N Protein
% of total protein % TPO 10Sa Tag pMP331 - - 5.8 11.3 7.6 pMP951 +
+, no SD 5.8 30.9 2.5 pMP1217 + +, full SD 5.0 33.0 1.7 pMP1099 - -
6.1 17.8 3.3 pMP1099/ - - 8.2 21.2 4.2 pDR1 pMP1099/ - - 8.2 21.2
4.8 pDR3 pMP1086 + +, no SD 7.3 27.6 2.3 pMP1086/ + +, no SD 7.7
37.5 2.0 pDR1 pMP1086/ + +, no SD 8.5 32.0 2.9 pDR3 pMP1201 + +,
full SD 6.8 43.7 1.6
[0164] The data in Table 2 show that expressing TPO from a plasmid
with the .lamda.N anti-termination system results in a dramatic
decrease in 10Sa-tagged TPO, as reflected in the column labeled
10Sa Tag. The percentage of TPO present as full-length TPO also
increases with the .lamda.N anti-termination system (% TPO column
of Table 2).
[0165] It is noted that the control plasmid with the AP promoter
(pMP1099), but not with the trp promoter (pMP331), produced more %
TPO in the fermentor than in the shake flask (compare Table 1 to
Table 2). The fermentor conditions provide a more constant glucose
feed and controlled pH with a longer induction period than the
shake-flask conditions, so that more protein is produced in the
former, using the AP promoter. However, with the anti-termination
system, the results show a consistent improved trend in % TPO
whether the TPO is produced in the shake flasks or in the fermentor
and whether the promoter is trp or AP. Moreover, the 10Sa tag is
lower for the anti-termination system in all cases.
[0166] Further, it is clear from the data that co-expressing TPO
and either GreA or GreB with or without the .lamda.N
anti-termination system results in an increase in TPO production (%
of total protein column in Table 2). There is also an increase in
the percentage of TPO present as full-length TPO when either GreA
or GreB is co-expressed with TPO.
EXAMPLE 4
Effect of .lamda.N Anti-Termination System on FGF-5 Production
(Fermentor)
Materials and Methods:
Description and Construction of the FGF-5 Expression Plasmids
Plasmid pFGF5IT
[0167] The hFGF-5 E. coli expression plasmid, pFGF5IT, was
constructed from a basic backbone of pBR322 (Sutcliffe, Cold Spring
Harb Symp Quant Biol., 43: 77-90 (1978)). The trp promoter provides
the transcriptional sequence required for efficient expression of
the FGF-5 gene in E. coli (Yanofsky et al., Nucleic Acids Res., 9:
6647-6668 (1981)). Two Shine-Dalgarno sequences, the trp
Shine-Dalgarno and a second Shine-Dalgarno, facilitated the
translation of FGF-5 mRNA (Yanofsky et al., Nucleic Acids Res., 9:
6647-6668 (1981); Ringquist et al., Molecular Microbiol., 6:
1219-1229 (1992)). The coding sequence for mature FGF-5 (lacking
the wild-type signal sequence) is located downstream of the
promoter and Shine-Dalgarno sequences and is preceded by a
methionine initiation codon.
[0168] The vector used for the construction of pFGF5IT was
generated by isolating the largest fragment when pRelCIII was
digested with XbaI and BamHI. This vector contains the trp promoter
and trp Shine-Dalgarno sequence. The second fragment required for
this construction was isolated by first digesting pFGF5I with
HindIII followed by treatment with DNA Polymerase I (Klenow
fragment) to create a blunt end. This reaction was then digested
with XbaI, resulting in a fragment of about 770 bp with one sticky
end (XbaI) and one blunt end (HindIII Pol). This fragment contains
a Shine-Dalgarno sequence, an initiation methionine codon, and the
coding sequence for mature hFGF-5. The final fragment required for
this construction was isolated from pdh108. This StuI-BamHI
fragment of about 420 bp contains the sequence encoding the
.lamda.t.sub.o transcriptional terminator (Scholtissek et al.,
Nucleic Acids Res., 15 (7): 3185 (1987)) and approximately the
first 375 bp of pBR322 (Sutcliffe, supra). These three fragments
were ligated together as depicted in FIG. 21 for the construction
of pFGF5IT.
Plasmid pFGF5IT-AT
[0169] The plasmid pFGF5IT-AT simply places the coding sequence for
FGF-5 into a .lamda.N anti-termination expression plasmid. The
vector used for this construction was created by isolating the
largest fragment when pMP951 was digested with XbaI and BamHI. This
vector contains the trp promoter and the nut site (Boxes A+B). The
second fragment required for this construction was isolated
following digestion of pFGF5IT-PhoA with XbaI and HincII. The
plasmid pFGF5IT-PhoA is a derivative of pFGF5IT in which the trp
promoter is replaced by the AP promoter (Kikuchi et al., supra).
This approximately 810-bp fragment contains a Shine-Dalgarno
sequence, a methionine initiation codon, the coding sequence for
mature hFGF5, and the sequence for the .lamda.t.sub.0
transcriptional terminator. The final fragment required for the
ligation was isolated by digestion of pMP931 with SspI and BamHI.
The SspI digestion was only a partial digestion, resulting in a
fragment of approximately 900 bp. This last fragment contains the
tacII promoter (without a Shine-Dalgarno) followed downstream by
the coding sequence for .lamda.N protein. These three fragments
were ligated together as illustrated in FIG. 22 to yield the
plasmid pFGF5IT-AT.
Transformation
[0170] The strain 54C2 (E. coli W3110 fhuA (tonA) lon galE rpoHts
(htpRts) clpP lacIq) was transformed with each of pFGF5IT or
pFGF5IT-AT using standard procedures involving ampicillin.
Culture of Transformed Cells
[0171] The transformed cells were grown up in a fermentor under
conditions described in Example 2 for the trp plasmids pMP331 and
pMP951, except that the IPTG solution was used only for pFGF5IT-AT
and not for pFGF5IT. Whole cell lysates from the fermentation
samples were prepared for SDS-PAGE.
Results:
[0172] The FGF-5 intracellular expression plasmid pFGF5IT produced
a significant amount of full-length protein (FIG. 23A, Lane 2).
Despite this promising result, truncated FGF-5 species also
accumulated in addition to the full-length protein (FIG. 23B, Lane
2), and the majority of these species were 10Sa-tagged (FIG. 23C,
Lane 2). These results imply, without being limited to any one
theory, that premature transcription termination is a likely source
of the truncation problem.
[0173] To address this problem, the .lamda.N anti-termination
system was co-expressed with the FGF-5 gene. The production of
full-length FGF-5 was approximately equivalent for both plasmids,
with or without .lamda.N anti-termination (FIG. 23A, lanes 2 and
3). However, the accumulation of truncated species was reduced by
approximately 50% when the .lamda.N anti-termination system was
co-expressed with the FGF-5-encoding gene (FIGS. 23B and 23C). The
reduction of these truncated species not only allows for simplified
purification of the FGF-5 full-length protein, but minimizing the
production of these smaller FGF-5 fragments also leads to improved
efficiency in refolding, since the smaller species may contribute
to aggregation in the refolding reactions. In addition, any
truncated species that do refold and remain in solution may
complicate the assessment of bioactivity by interfering with the
binding of full-length protein to the receptor. Thus, it is
advantageous to reduce the level of premature transcription
termination to prevent these potential problems from arising.
EXAMPLE 5
Effects of .lamda.N Anti-Termination and GreA/B on FGF-5 Production
(Shake-Flask)
Materials and Methods:
Shake Flask Experiments with FGF-5 and GreA or GreB
Co-Expression:
[0174] The strain 59B9 {W3110 fhuA.DELTA. (tonA.DELTA.) lon.DELTA.
galE rpoHts (htpRts) .DELTA.clpP lacIq .DELTA.ompT
.DELTA.(nmpc-fepE) .DELTA.lacY} was transformed with pFGF5IT-PhoA
(described in Example 4) or pFGF5IT-PhoAAT, either alone or in
combination with pDR1 or pDR3, each of which is described above.
The plasmid pFGF5IT-PhoAAT is a derivative of pFGF5IT in which the
same anti-termination element is present as in pFGF5IT-AT described
in Example 4, and in which the trp promoter is replaced by the AP
promoter (Kikuchi et al., supra).
Culture of the Transformed Cells
[0175] The transformed cells were grown in LB media with ampicillin
and kanamycin (when co-transformed with pDR1 and pDR3 only) at
30.degree. C. with shaking overnight and then diluted 50-fold into
C.R.A.P. medium containing the appropriate antibiotics and grown at
30.degree. C. with shaking. Transformants were grown in this medium
until they reached an OD550 of 1-2, at which time IPTG was added (1
mM, final concentration) to all of the cultures except for the one
containing pFGF5IT-PhoA alone. All cultures were grown for a total
of 24 hours. Samples were then removed and prepared for
SDS-PAGE.
Quantitation of 10Sa-Tagged and Full-Length FGF-5:
[0176] Samples from the shake flask cultures described above were
prepared and run on SDS-PAGE, transferred to nitrocellulose, and
probed with a polyclonal antibody raised against either FGF-5
(R&D Systems) or the 10Sa peptide. The blots were then scanned
using an optically enhanced laser densitometer (PDI, Inc., model
325oe). The peak areas for the full-length FGF-5, as well as the
other FGF-5 species that cross-react with the FGF-5 polyclonal
antibody, were determined.
Results:
[0177] The results from these analyses are shown in Table 3. The
ratio of full-length FGF-5 to all of the FGF-5 species was
calculated and is reported as % FGF-5. In addition, the total peak
area detected on the blot probed with the polyclonal antibody
raised to the 10Sa peptide was also calculated and is reported as
10Sa tag. TABLE-US-00013 TABLE 3 Effect of Plasmid Construct on
FGF-5 Accumulation and 10Sa Tagging Plasmid(s) % FGF-5 10Sa Tag
pFGF5IT-PhoA 15 10.6 pFGF5IT-PhoA/ 16 11.3 pDR1 pFGF5IT-PhoA/ 21
7.6 pDR3 pFGF5IT-PhoAAT 40 3.0 pFGF5IT-PhoAAT/ 52 1.7 pDR1
pFGF5IT-PhoAAT/ 45 3.4 pDR3
[0178] The data in Table 3 show that expressing FGF-5 from a
plasmid with the .lamda.N anti-termination system results in a
dramatic decrease in 10Sa-tagged FGF-5, as demonstrated by the scan
data for 10Sa-tagged proteins in Table 3. The percentage of FGF-5
present as full-length FGF-5 also increases with the .lamda.N
anti-termination system.
[0179] Further, it is clear from the data that co-expressing FGF-5
with either GreA or GreB with or without the .lamda.N
anti-termination system results in an increase in the percentage of
FGF-5 present as full-length FGF-5, as shown by the data in the
column labeled % FGF-5.
Sequence CWU 1
1
19 1 80 DNA Artificial Sequence Expression construct 1 ttaactagta
cgcaacgctc ttacacattc cagccctgaa aaagggcaaa 50 gttcacgtaa
aaaggatatc tagaattatg 80 2 114 DNA Artificial Sequence Expression
construct 2 tatagtcgct ttgtttttat tttttaatgt atttgtaact agtacgcaac
50 gctcttacac attccagccc tgaaaaaggg caaagttcac gtaaaaagga 100
tatctagaat tatg 114 3 5 PRT Artificial Sequence Fragment of phage
lambda N 3 Met Asp Ala Gln Thr 5 4 56 DNA Artificial Sequence E.
coli and phage lambda N fragment fusion 4 tttaatgtgt ggaattgtga
gcggataaca attaagcttt tatggatgca 50 caaaca 56 5 68 DNA Artificial
Sequence E. coli and phage lambda N fragment fusion 5 tttaatgtgt
ggaattgtga gcggataaca attaagctta ggattctaga 50 attatggatg cacaaaca
68 6 4 PRT Homo sapiens 6 Ile Glu Pro Arg 7 60 DNA Artificial
Sequence Fragment for plasmid construction 7 ctagttaact agtacgcatt
ccagccctga aaaagggcaa agttcacgta 50 aaaaggatat 60 8 60 DNA
Artificial Sequence Fragment for plasmid construction 8 ctagatatcc
tttttacgtg aactttgccc tttttcaggg ctggaatgcg 50 tactagttaa 60 9 40
DNA Artificial Sequence Fragment for plasmid construction 9
ctgtctcagg aagggtaagc ttttatggat gcacaaacac 40 10 47 DNA Artificial
Sequence Fragment for plasmid construction 10 cggcgtgttt gtgcatccat
aaaagcttac ccttcctgag acagatt 47 11 35 DNA Artificial Sequence
Fragment for plasmid construction 11 agcttaggat tctagaatta
tggatgcaca aacac 35 12 35 DNA Artificial Sequence Fragment for
plasmid construction 12 cggcgtgttt gtgcatccat aattctagaa tccta 35
13 73 DNA Artificial Sequence Fragment for plasmid construction 13
ctagttaact agtacgcaac gctcttacac attccagccc tgaaaaaggg 50
caaagttcac gtaaaaagga tat 73 14 73 DNA Artificial Sequence Fragment
for plasmid construction 14 ctagatatcc tttttacgtg aactttgccc
tttttcaggg ctggaatgtg 50 taagagcgtt gcgtactagt taa 73 15 48 DNA
Artificial Sequence Primer 15 ccccccccct ctagaaaaat gaaaactcct
ctggtaacgc gggaaggg 48 16 39 DNA Artificial Sequence Primer 16
cccccccccc tgcagttacg gtttcacgta ctcgatagc 39 17 44 DNA Artificial
Sequence Primer 17 ccccccccct ctagaattct atgcaagcta ttccgatgac ctta
44 18 36 DNA Artificial Sequence Primer 18 cccccccccc tgcagttaca
ggtattccac cttaat 36 19 12 PRT Artificial Sequence Peptide for
generating antibodies 19 Cys Ala Ala Asn Asp Glu Asn Tyr Ala Leu
Ala Ala 5 10
* * * * *