Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-03-01
2001-06-05
Degen, Nancy (Department: 1805)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S173300, C536S023700, C536S024100
Reexamination Certificate
active
06242177
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to signal sequences for the secretion of heterologous polypeptides from bacteria.
DESCRIPTION OF BACKGROUND AND RELATED ART
Secretion of heterologous polypeptides into the periplasmic space of
E. coli
and other prokaryotes or into their culture media is subject to a variety of parameters. Typically, vectors for secretion of a polypeptide of interest are engineered to position DNA encoding a secretory signal sequence 5′ to the DNA encoding the polypeptide of interest. Two major recurring problems plague the secretion of such polypeptides. First, the signal sequence is often incompletely processed or removed, and second, the amount of polypeptide secreted is often low or undetectable. Attempts to overcome these problems fall into three major areas: trying several different signal sequences, mutating the amino acid sequence of the signal sequence, and altering the secretory pathway within the host bacterium.
A number of signal sequences are available for the first approach in overcoming secretion problems. Watson (
Nucleic Acids Research
12: 5145-5164 (1984)) discloses a compilation of signal sequences. U.S. Pat. No. 4,963,495 discloses the expression and secretion of mature eukaryotic protein in the periplasmic space of a host organism using a prokaryotic secretion signal sequence DNA linked at its 3′ end to the 5′ end of the DNA encoding the mature protein. In particular, the DNA encoding
E. coli
enterotoxin signals, especially STII, are preferred. Chang et al. (
Gene
55:189-196 (1987)) discloses the use of the STII signal sequence to secrete hGH in
E. coli.
Gray et al. (
Gene
39:247-245 (1985)) disclose the use of the natural signal sequence of human growth hormone and the use of the
E. coli
alkaline phosphatase promoter and signal sequence for the secretion of human growth hormone in
E. coli.
Wong et al. (
Gene
68:193-203 (1988)) disclose the secretion of insulin-like growth factor 1 (IGF-1) fused to LamB and OmpF secretion leader sequences in
E. coli
, and the enhancement of processing efficiency of these signal sequences in the presence of a prlA4 mutation. Fujimoto et al. (
J. Biotech.
8:77-86 (1988)) disclose the use of four different
E. coli
enterotoxin signal sequences, STI, STII, LT-A, and LT-B for the secretion of human epidermal growth factor (hEGF) in
E. coli.
Denefle et al. (
Gene
85: 499-510 (1989)) disclose the use of OmpA and PhoA signal peptides for the secretion of mature human interleukin 1&bgr;.
Mutagenesis of the signal sequence has, in general, not been especially helpful in overcoming secretion problems. For example, Morioka-Fujimoto et al. (
J. Biol. Chem.
266:1728-1732 (1991)) disclose amino acid changes in the LTA signal sequence that increased the amount of human epidermal growth factor secreted in
E. coli
. Goldstein et al. (
J. Bact.
172:1225-1231 (1990)) disclose amino acid substitution in the hydrophobic region of OmpA effected secretion of nuclease A but not TEM &bgr;-lactamase. Matteucci et al. (
Biotech.
4:51-55 (1986)) disclose mutations in the signal sequence of human growth hormone that enhance secretion of hGH. Lehnhardt et al. (
J. Biol. Chem.
262:1716-1719 (1987) disclose the effect of deletion mutations in OmpA signal peptide on secretion of nuclease A and TEM &bgr;-lactamase.
Finally, attempts at improving heterologous secretion in
E. coli
by modulating host machinery has so far shown limited improvement in overcoming secretion problems. For example, van Dijl et al. (
Mol. Gen. Genet.
227:40-48 (1991)) disclose the effects of overproduction of the
E. coli
signal peptidase I (SPase I) on the processing of precursors. Klein et al. (
Protein Engineering
5:511-517 (1992) disclose that mutagenesis of the LamB signal sequence had little effect on secretion of bovine somatotropin, and that secretion properties of bovine somatotropin appear to be determined by the mature protein rather than by changes in the signal sequence. Perez-Perez et al. (
Bio/Technology
12:179-180 (1994)) disclose that providing an
E. coli
host with additional copies of prlA4 (secY allele) and secE genes, which encode the major components of the “translocator”, i.e., the molecular apparatus that physically moves proteins across the membrane, increased the ratio of mature to precursor hIL-6 from 1.2 to 10.8. U.S. Pat. No. 5,232,840 discloses novel ribosome binding sites useful in enhancing protein production in bacteria through enhanced and/or more efficient translation. U.S. Pat. No. 5,082,783 discloses improved secretion of heterologous proteins by hosts such as yeasts by using promoters of at most intermediate strength with heterologous DNA secretion signal sequences. European Patent Application No. 84308928.5, filed Dec. 19, 1984, discloses promoter-ribosome binding site expression elements of general utility for high level heterologous gene expression.
The instant invention discloses the unexpected result that altered translation initiation regions with reduced translational strength provided essentially complete processing and high levels of secretion of a polypeptide of interest as compared to wild type sequences, and that many mammalian polypeptides require a narrow range of translation levels to achieve maximum secretion. A set of vectors with variant translation initiation regions provides a range of translational strengths for optimizing secretion of a polypeptide of interest.
SUMMARY OF THE INVENTION
One aspect of the invention is a method of optimizing secretion of a heterologous polypeptide of interest in a cell comprising comparing the levels of expression of the polypeptide under control of a set of nucleic acid variants of a translation initiation region, wherein the set of variants represents a range of translational strengths, and determining the optimal translational strength for production of mature polypeptide, wherein the optimal translational strength is less than the translational strength of the wild-type translation initiation region.
In a further aspect of the invention the variants are signal sequence variants, especially variants of the STII signal sequence.
REFERENCES:
patent: 4963495 (1990-10-01), Chang et al.
patent: 5232840 (1993-08-01), Olins
patent: 177343 (1986-04-01), None
Chang et al., “High-level secretion of human growth hormone byEscherichia coli”Gene55:189-196 (1987).
Denefle et al., “Heterologous protein export inEscherichia coli: influence of bacterial signal peptides on the export of human interleukin 1&bgr;”Gene85:499-510 (1989).
Fujimoto et al., “Expression and secretion of human epidermal growth factor byEscherichia coliusing enterotoxin signal sequences”J. Biotech. 8:77-86 (1988).
Goldstein et al., “Enhancement of Protein Translocation across the Membrane by Specific Mutations in the Hydrophobic Region of the Signal Peptide”J. Bact.172(3):1225-1231 (Mar. 1990).
Gray et al., “Periplasmic production of correctly processed human growth hormone inEscherichia coli: natural and bacterial signal sequences are interchangeable”Gene39:247-254 (1985).
Klein et al., “Effects of signal peptide changes on the secretion of bovine somatotropin (bST) fromEscherichia coli”Protein Engineering5(6):511-517 91992).
Lehnhardt et al., “The Differential Effect on Two Hybrid Proteins of Deletion Mutations within the Hydrophobic Region of theEscherichia coliOmpA Signal Peptide”Journal of Biological Chemistry262(4):1716-1719 (Feb. 5, 1987).
Matteucci et al., “Alkaline Phosphatase Fusions: A Tag to Identify Mutations that Result in Increased Expression of Secreted Human Growth Hormone fromE. Coli”Biotechnology4:51-55 (Jan. 1986).
Morioka-Fujimoto et al., “Modified Enterotoxin Signal Sequences Increase Secretion Level of the Recombinant Human Epidermal Growth Factor inEscherichia coli”Journal of Biological Chemistry266(3) :1728-1732 (1991).
Perez-Perez et al., “Increasing the Efficiency of Protein Export inEscherichia coli”Bio/Technology12:178-180 (Feb. 12, 1994).
van Dijl et al., “Signal peptidase I overproduction results in increased efficiencies of export and ma
Simmons Laura C.
Yansura Daniel G.
Degen Nancy
Flehr Hohbach Test Albritton & Herbert LLP
Genentech Inc.
Hasak Janet
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