Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-01-26
2001-01-30
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06180391
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to new and improved expression vector systems capable of expressing exogenous genes, including toxic genes, in
E. coli
and other host cells. More specifically, the invention relates to highly efficient, tightly regulated expression vector systems which utilize the bacteriophage MS2 coat protein (MS2CP) and MS2 recognition site to control translation of the mRNA for any cloned gene in a host cell. In addition, the invention relates to expression vector systems which utilize transcriptional control proteins, e.g., motA and asiA of bacteriophage T4, to regulate transcription and provide a staged inducible promoter system which is much less complicated and much more versatile than the non-staged systems previously described.
BACKGROUND OF THE INVENTION
Prokaryotic cells have become the system of choice for expression of cloned genes encoding eukaryotic and prokaryotic polypeptides, and numerous expression systems exist for expression of gene products in bacteria. The expression of genes in
E. coli
has become established as a key technique in the understanding of molecular processes and
E. coli
expression systems have become a standard and popular method for the production and large-scale purification of exogenous proteins. Importantly, the technology has provided a source of proteins in a quantity and quality that was previously difficult, or impossible, to achieve through isolation from natural sources.
A number of patented expression systems for use in bacterial hosts have been described. In some instances, the expression systems described relate to generalized expression systems, while in other instances, the patented expression system is designed to allow relatively tight regulation, or is individually tailored for expression of a particular protein. Examples of such patents include U.S. Pat. No. 4,767,708 (construction of recombinant vector containing a cloned bacterial DNA polymerase I under the control of a positively regulated foreign promoter); U.S. Pat. No. 4,503,142 (construction of a class of cloning and expression vectors based on the use of the lac promoter/operator of
E. coli
); and U.S. Pat. No. 4,578,355 (use of the P
L
promoter of bacteriophage lambda to construct high level expression vectors).
Another widely used expression system, referred to as the pET vector expression system, makes use of the powerful T7 RNA polymerase to transcribe genes of interest; Studier et al.,
J. Mol. Biol.,
189:113-130 (1986). T7 RNA polymerase is highly selective for it own promoters, and no T7 promoters are known to be present in the DNA of
E. coli
. T7 uses its highly selective polymerase to direct transcription to its own DNA rather than to host DNA during infection, and a relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Moreover, T7 RNA polymerase is at least 5 times more active than
E. coli
polymerases; Id. Many heterologous proteins have been successfully expressed in high yields using the pET system; see, e.g. Dietrich et al.,
Eur. J. Biochem,
201:399-407 (1991); Lathrop et al.,
Protein Expr. Purif.,
3:512-517 (1992); Aukhil et al.,
J. Biol. Chem.,
268:2542-2553 (1993).
Unfortunately, these and other reported expression systems still suffer, to a greater or lesser degree, from such things as: 1) uninduced expression resulting from promoter leakiness; 2) inability to control expression to the extent necessary when the gene product will kill or otherwise seriously damage the host cell if expressed (more typically associated with expression of eukaryotic genes in bacteria); 3) the need to rely for induction of expression on either the host's biochemical responses or expensive or otherwise inadequate induction mechanisms; 4) inability to express gene of interest at sufficient levels; and 5) plasmid instability. Until such problems are adequately addressed and successfully overcome, the full potential of such expression systems will not be fully appreciated or utilized.
All known inducible promoter systems have a residual level of activity or “leakiness” which leads to the inappropriate transcription and expression of the gene being cloned under the control of the promoter. In most cases, this is not a problem because the gene product being produced is well tolerated by the cell, i.e., the gene product is non-toxic. However, in instances where the gene product being produced is toxic or even lethal to the cell, even these small amounts of expression can be detrimental. In fact, there are certain toxic genes that have been characterized as “unclonable” because they are unstable in any cloning vector.
Studier, F. W.,
J. Mol. Biol.,
219:37-44 (1991) addressed the problem of leaky expression in the pET vectors by developing the Lys series of vectors. These vectors harbor the gene for T7 lysozyme, which binds the T7 RNA polymerase present in the uninduced cell and prevents it from transcribing target genes. Dubendorff and Studier,
J. Mol. Biol.,
219:45-59 (1991) addressed the problem by providing additional copies of lac operon control elements in the pET plasmids. Brown et al.,
Gene,
127:99-103 (1993) used induction by infection with a mutant T7 phage to successfully clone the toxic POL3 gene. Unfortunately, these and other reported systems fail to completely alleviate the leakage problem or suffer from the problems of plasmid instability and cell lysis associated with high levels of lysozyme and/or phage infection.
The present invention addresses and overcomes the problem of promoter leakiness by utilizing the translational repression capabilities of the bacteriophage MS2 coat protein. Bacteriophage MS2 is an RNA-containing phage with a fairly simple life cycle; Van Duin, J.,
Single
-
Stranded RNA Bacteriophages in The Bacteriophages
, Chapter 4, pgs. 117-167, Plenum Press, New York (1988). The RNA encodes four genes, one of which is a replicase gene that copies the genome, another of which is the multifunctional coat protein gene. As the concentration of the coat protein increases in the cell, it forms a dimer and binds to a hairpin structure consisting of the ribosome binding site and ATG of the replicase gene. This binding then inhibits further translation from the replicase gene and effectively shifts the life cycle to the packaging of the genome.
The sequence of the MS2 coat protein has been published; Fiers et al.,
Nature,
260:500-507 (1976), mutants of the coat protein have been isolated and characterized; Peabody & Ely,
Nucleic Acids Research,
20(7):1649-1655 (1992), and construction of a two-plasmid system for genetic analysis of the translational repressor activity of coat protein has been described; Peabody, D.,
Journ. of Biol. Chem.,
265(10):5684-5689 (1990).
Bacteriophage MS2 is not the only phage known to have proteins that bind messenger RNA and inhibit translation. For example, the RegA protein of bacteriophage T4 and the gene V protein of M13 are two other such proteins. However, a key difference between the RegA protein, gene V protein, and the MS2 coat protein relates to the placement of their recognition sites relative to the start site of the gene. For example, the RegA protein's recognition site is comprised of sequences in the coding region of the gene, thus limiting the genes that may be controlled by it. The gene V protein recognizes a sequence upstream of the ribosome binding sequence and thus may still allow a low level of translational initiation. For MS2, the binding site does not cover coding regions of the gene but does include the ribosome binding sequence and initiation codon. The MS2 system thus allows for the tightest possible repression, while still allowing for the site to be used universally.
The specific use of the MS2CP and MS2 recognition site will be described in more detail in the examples provided herein. As demonstrated by those examples, this unique use of the MS2 system allows one of ordinary skill in the art to eff
Amgen Inc.
Crandall Craig A.
Levy Ron K.
Odre Steven M.
Schwartzman Robert A.
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