Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1992-12-18
2001-02-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C536S024100, C435S069100
Reexamination Certificate
active
06194200
ABSTRACT:
INTRODUCTION
1. Technical Field
This invention relates to compositions and methods for preparation of novel polypeptides, in particular fusion polypeptides, using recombinant DNA techniques.
2. Background
The advent of genetic engineering brought with it the promise of easy production of large quantities of a variety of peptides. However, this promise has not been fully realized for a number of reasons. For example, in many instances where the peptide has been produced and retained in the cytoplasm of the host organism, inclusion bodies have resulted requiring denaturation and renaturation of the protein, frequently with only partial or little success. In other instances, the peptide has been substantially degraded so that not only are yields low, but also complicated mixtures are obtained, which are difficult to separate.
As a potential solution to these difficulties, the possibility of obtaining secretion of a desired peptide into the nutrient medium has been investigated. Obtaining secretion of the desired protein has met with limited success in the past, since not all proteins are capable of being secreted by the host cells which have been employed. Moreover, even when secreted, the processing of the peptide by the host cell may result in a product which differs from the composition and/or conformation of the desired polypeptide and the yields of protein have been less than expected. There is, therefore, a substantial interest in developing systems for the efficient and economic production of active peptides where the desired polypeptide can accumulate in the host cell without degradation and can either be secreted in an active conformation or conveniently processed and renatured to a functional state.
SUMMARY OF THE INVENTION
Expression cassettes, and methods for their preparation and use are described, which provide for enhanced expression and production of an active gene product. The expression cassettes include efficient transcriptional and translational initiation and termination regulatory regions appropriate for the host cell to provide for expression of a desired polypeptide. The expression cassette preferably further includes, as appropriate for the host cell, a leader sequence for expression under the transcriptional and translational regulation of the regulatory region, sequences providing for enzymatic or chemical cleavage sites for cleavage of the leader peptide from mature polypeptide, and regulatory sequences which allow the time of expression of the gene of interest to be modulated. The expression cassettes are introduced into a host cell under conditions whereby the resulting transformants stably maintain the expression cassette. Naturally occuring DNA and synthetic genes may be employed for the production of a polypetide of interest.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, expression cassettes are provided which, when inserted into a host cell, allow for the preparation of a polypeptide of interest which has enhanced stability and is either secreted in an active conformation or may be conveniently processed and renatured to an active state.
To obtain increased expression of a polypeptide of interest in a host cell, the nucleotides encoding the N-terminal amino acids of the polypeptide of interest are modified within the constraints of codon degeneracy to mimic those of the natural gene sequence found with the Shine-Dalgarno sequence used in the expression cassette. The expression cassette thus will have the following general structure:
P--S.D.--met--G
1
wherein
P comprises a promoter sequence including the regulatory regions occurring at about −35 and −10 nucleotides upstream from the start of the RNA chain and may also include regulatory sequences allowing for the induction of regulation;
S.D. comprises a Shine-Dalgarno sequence;
met comprises a codon for the initiating methionine of the polypeptide of interest; and
G
1
comprises the gene for the polypeptide of interest wherein the first 7 to 30 codons of the gene have been modified wherever possible, using codon degeneracy to approximate the nucleotide sequence of the natural gene which would follow the Shine-Dalgarno sequence used in the expression cassette.
As an alternate means of obtaining increased expression of the polypeptide of interest from the host cell, the polypeptide can be expressed as a fusion protein by including in the expression cassette a DNA sequence encoding a leader sequence peptide joined in reading frame upstream from the gene of interest. Expressing the polypeptide of interest as a fusion protein can result in up to 30% or more of the protein produced by the host cell being the polypeptide of interest. The expression cassette for expressing a fusion protein will thus have the following basic structure:
P--S.D.--met--L--G
wherein:
P, S.D. and met have the meaning described above;
G comprises a gene for the polypeptide of interest; and
L comprises a DNA sequence encoding a leader peptide which may be an N-terminal sequence from any bacterial or bacteriophage gene, but generally is from a highly expressed gene; an amino acid sequence containing large numbers of hydrophobic amino acid residues; or an amino acid sequence containing large numbers of hydrophilic amino acid residues. When L comprises a hydrophobic amino acid sequence, this sequence will preferably also function as a signal sequence, allowing secretion of the polypeptide of interest from the host cell and cleavage of the signal sequence from the polypeptide. The DNA sequence coding for L may also be modified wherever possible, using codon degeneracy to approximate the nucleotide sequence of the natural gene which would follow the Shine-Dalgarno sequence used in the expression cassette.
The expression cassette described above provides for a fused expression product comprising the leader peptide and the polypeptide of interest. If it is desired to obtain the polypeptide of interest alone and there is no convenient cleavage site, e.g. as provided by a natural signal sequence, a cleavage site may be provided for by joining at least one codon encoding a cleavage site in reading frame upstream from the gene of interest. The cassette will thus have the following structure:
P--S.D.--met--L--C--G
wherein P, S.D., met, L and G have the meaning described above and
C comprises at least one codon providing for a chemical or enzymatic cleavage site.
To stabilize the mRNA and to provide for higher levels of expression of a desired polypeptide, a transcriptional termination region (T) can be included in the expression cassette downstream from the gene of interest. An example of an expression cassette comprising T is as follows:
P--S.D.--met--G--T
although T may be included in any of the expression cassettes as described above.
Construction of Expression Cassettes
Design of an expression system to yield high levels of gene product must take into consideration not only the particular regions of a gene which have been determined to influence expression but also how these regions (and thus their sequences) influence each other. Where possible, choice of appropriate regulatory sequences will take into account the various factors which affect expression. Different genes have evolved a combination of all of these factors to yield a particular rate of expression; thus highly expressed genes can be considered useful models.
In terms of transcriptional regulation, the amount and stability of messenger RNA are important factors which influence the expression of gene products. The amount of mRNA is determined by the copy number of a particular gene, the relative efficiency of its promoter and the factors which regulate the promoter, such as enhancers or repressors. Initiation is believed to occur in the region just upstream of the beginning of the coding sequence.
The promoter in prokaryotic cells comprises nucleotide sequences which can affect the efficiency of transcription. These sequences include the regulatory regions at about −35 and −10 nucleotides from the start of the RNA
Bruce A. Gregory
Rose Timothy M.
Guzo David
Oncogen
Pennie & Edmonds LLP
LandOfFree
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