Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
2000-01-31
2002-04-02
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C702S019000, C702S030000, C702S032000, C540S456000, C540S460000
Reexamination Certificate
active
06366860
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is synthetic nucleic acid sequences for improved amplification and expression in a host organism, and methods of creating them.
It has been a goal of biotechnology to promote the expression of cloned genes for analysis of gene structure and function and also for commercial-scale synthesis of desirable gene products. DNA cloning methods have enabled the genetic modification of bacteria and unicellular eukaryotes to produce heterologous gene products. In principle, the genes may originate from almost any source, including other bacteria, animal cells or plant cells. Although this expression of heterologous genes is a function of a variety of complex factors, maximizing the expression of cloned sequences has been under intense and rapid development. Plasmid and viral vectors have been developed in both prokaryotes and eukaryotes that enhance the level of expression of cloned genes. In some cases the vector itself contains the regulatory elements controlling the expression of genes which are not normally expressed in the host cell so that a high level of expression of heterologous genes can be obtained.
Several problems exist, however, in the expression of many proteins across phyla and even across species. Post-translational handling and modification of expressed proteins by the host cell often does not mimic that of the heterologous gene's own cell type. Frequently, even if the protein is expressed in a useful form, heterologous genes are poorly expressed. Low yields of expressed protein may make manufacture of commercially useful quantities impossible or prohibitively costly. Vectors designed to enhance expression are not able to overcome some expression problems if the regulatory elements of the vector are not the constraint on robust expression. Other cellular or translational constraints are at issue.
Genes encoding poorly expressed proteins are often themselves difficult to clone and amplify as well. This is due to secondary structure inherent in the gene, for example caused by high G-C content. Some methods have been used to reduce these difficulties, such as the use of DMSO or betaine to bring G-C and A-T melting behaviors more into alignment, or the use of ammonium sulfate (hydrogen binding cations) to destabilize G-C bonding during PCR. The problem with these methods is that the effects of the additives are concentration dependent, so variations in template size and G-C content mean lengthy optimization procedures. Additionally, these steps do nothing to facilitate subsequent expression of the nucleic acid once it has been cloned.
The frequency of particular codon usage in
Escherichia coli
and other enteric bacteria has long been known, and it has been hypothesized that replacement of certain rare codons encoding a particular amino acid in a heterologous gene with a codon that is more commonly used by such bacteria would enhance expression (see, e.g., Kane,
Curr Opin Biotechnol
6:494-500 (1995) and Zahn,
J. Bacteriol.,
178:2926-2933 (1996)). This is based on the theory that rare codons have only a few tRNAs per cell and that transcription of heterologous sequences having numerous occurrences of these rare codons is limited by too few available tRNAs for those codons. However, simple replacement of rare codons does not reliably improve expression of heterologous genes, and no broadly applicable method exists to select which codon changes are best to increase expression of heterologous sequences. Further, it is not known in detail how codon usage is related to expression level.
Bacterial gene products are commonly used as research and assay reagents, and various microbial enzymes increasingly are finding applications as industrial catalysts (see, for example, Rozzell, J. D., “Commercial Scale Biocatalysis: Myths and Realities,”
Bioorganic and Medicinal Chemistry,
7:2253-2261 (1999), herein incorporated by reference). Some have substantial commercial value. Examples include heat-stable Taq polymerase from
Thermus aquaticus
, restriction enzymes such as Eco RI from
E. coli
, lipase from
Pseudomonas cepacia
, &bgr;-amylase from Bacillus sp., penicillin amidase from
E. coil
and Bacillus sp., glucose isomerase from the genus Streptomyces, and dehalogenase from
Pseudomonas putida
. Genes from bacteria may express easily in commercially useful host strains, but many do not. In particular, genes from bacteria having significantly different codon preferences from enteric bacteria, including but not limited to filamentous bacteria such as streptomycetes and various strains of the genus Bacillus, Pseudomonas, and the like can be difficult to express abundantly in enteric bacteria such as
E. coli
. An example of a Pseudomonas gene that is difficult to express in
E. coli
is the enzyme methionine gamma-lyase, useful for the assay of L-homocysteine and/or L-methionine as described in U.S. Pat. No. 5,885,767 (herein incorporated by reference). This assay is particularly useful in the diagnosis and treatment of homocystinuria, a serious genetic disorder characterized by an accumulation of elevated levels of L-homocysteine, L-methionine and metabolites of L-homocysteine in the blood and urine. Homocystinuria is more fully described in Mudd et al., “Disorders of transsulfuration,” In: Scriver et al., eds.,
The Metabolic and Molecular Basis of Inherited Disease
, McGraw-Hill Co., New York, 7
th
Edition, 1995, pp. 1279-1327 (herein incorporated by reference). In developing an assay for the accurate quantitation of L-homocysteine and L-methionine according to the methods described in U.S. Pat. No. 5,885,767, obtaining large amounts of methionine gamma-lyase is necessary. However, this Pseudomonas gene contains a number of codons that are less commonly found in genes of desirable bacterial hosts for expression such as
E. coli.
Because plasmid vectors designed to enhance expression with a variety of promotors or other regulatory elements often do not resolve the difficulty in expressing certain genes, and because no systematic approach exists for codon replacement to aid amplification of nucleic acids or their expression, there is clearly a need for an improved method for amplification and expression of genes, including genes from various bacteria such as streptomycetes, Bacillus, Pseudomonas and the like introduced into enteric bacterial hosts such as
E. coli.
SUMMARY OF THE INVENTION
In one embodiment, the invention is directed to a method of making a synthetic nucleic acid sequence. The method comprises providing a starting nucleic acid sequence, which optionally encodes an amino acid sequence, and determining the predicted &Dgr;G
folding
of the sequence. The starting nucleic acid sequence can be a naturally occurring sequence or a non-naturally occurring sequence. The starting nucleic acid sequence is modified by replacing at least one codon from the starting nucleic acid sequence with a different corresponding codon to provide a modified nucleic acid sequence. As used herein, “codon” generally refers to a nucleotide triplet which codes for an amino acid or translational signal (e.g., a stop codon), but can also mean a nucleotide triplet which does not encode an amino acid, as would be the case if the synthetic or modified nucleic acid sequence does not encode a protein (e.g., upstream regulatory elements, signaling sequences such as promotors, etc.). As used herein, a “different corresponding codon” refers to a codon which does not have the identical nucleotide sequence, but which encodes the identical amino acid. The predicted &Dgr;G
folding
of the modified nucleic acid sequence is determined and compared with the &Dgr;G
folding
of the starting nucleic acid sequence. In accordance with the invention, the predicted &Dgr;G
folding
of the starting nucleic acid sequence can be determined before or after the modified starting nucleic acid is provided.
Thereafter, it is determined whether the &Dgr;G
folding
of the modified nucleic acid sequence is increased relative to the &Dgr;G
folding
of the starting nuc
Bui Peter
Rozzell, Jr. J. David
Biocatalytics, Inc.
Brusca John S.
Kim Young
LandOfFree
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