Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-10-05
2001-02-13
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S199000, C435S320100
Reexamination Certificate
active
06187589
ABSTRACT:
The invention relates to a vector system for cloning.
As a rule, common vectors which are used for cloning in prokaryotic systems contain the following features: a selection gene, e.g. the gene encoding resistance to ampicillin, a marker gene which makes it possible, e.g. on the basis of a color reaction, as in the case of the lacZ gene, to distinguish vectors with and without insert, and, especially, an origin of replication. The reader is also referred to the following documents with regard to the state of the art:
a) EP 0 532 043 A2,
b) EP 0 466 332 A2,
c) EP 0 293 249 A1,
d) GB 22 12 160 A and
e) U.S. Pat. No. 51 96 524.
The vector system described in publication c consists of a sequence of nucleotides which codes for the expression of a fusion protein. However, the foreign protein sequence in this case is linked directly to the sequence for an enzyme and is not separated by additional structures during the cloning process.
In systems which are based on phages rather than plasmids, further genetic elements are included which are important for the functions of the life cycle of the phage. Cleavage sites which occur only a few times in the vector, preferably only once, are normally used in these systems for inserting a foreign sequence into a marker gene. A series of such cleavage sites is normally arranged in a so-called multiple cloning site. Further elements, apart from this multiple cloning site, are included in some vectors which are used for expressing foreign proteins. In some genes, use is made, for this purpose, of a sequence which results, together with the insert to be cloned, in a fusion protein which then has an affinity for, e.g., maltose residues or nickel chelates. In some cases, these residues are joined by a recognition sequence for a proteinase, e.g. factor Xa. After purification by means of affinity chromatography has been effected, these proteinase cleavage sites make it possible to cleave the fusion protein into the affinity moiety and the foreign protein which is actually to be expressed. Since, however, this proteinase cleavage site is as a rule followed by a multiple cloning site sequence into which the sequence to be expressed has been cloned, additional, frequently unwanted amino acids remain on the foreign protein to be expressed after the fusion protein has been subjected to the processing with proteinase.
Most foreign protein sequences which are to be expressed are present in a nucleic acid environment which does not allow direct cloning directly after an endoproteinase recognition sequence to be effected efficiently using current cloning strategies. Using a multiple cloning site immediately after the endoproteinase recognition sequence (i.e. 3′ of the endoproteinase recognition sequence) markedly facilitates cloning of foreign protein sequences which are to be expressed. In this connection, it is disadvantageous that, at the protein level, it is not the foreign protein to be expressed which is recovered following digestion with the enzyme which recognizes the corresponding protein sequence; instead, that which is recovered is a fusion protein which comprises the foreign protein sequence to be expressed together with additional amino acids which are encoded by the residues of the multiple cloning site.
The object of the invention is to provide a novel vector system for cloning foreign proteins which avoids the disadvantages of the state of the art. This object is achieved by the features of claims
1
and
10
. Advantageous embodiments ensue from the features of claims
2
-
9
and
11
.
Use of the cloning vector system which is presented here now makes it possible, in a subsequent digestion and religation step, to bring the foreign protein sequence to be expressed directly up against an endoproteinase recognition sequence. This ensures that, after digestion with the enzyme which recognizes the corresponding protein sequence, the foreign protein sequence to be expressed is released, without any further components, from an expressed fusion protein.
A particular advantage is also to be seen in the fact that it is possible to select the reading frame at will during the process which is presented here. This makes it possible not only to exactly position the foreign gene component to be expressed but also to define the start of the fusion gene component. The following strategy was selective for preventing, in a simple but nevertheless highly selective manner, the unwanted expression, occasioned by the cloning procedure, of additional peptide components on the foreign gene to be expressed:
Use was made of the recognition sequence for the enzyme BcgI in order to clone it within the multiple cloning site, into which the foreign sequence can subsequently be inserted using any arbitrary enzymes. BcgI is one of the enzymes, so far the only one which is commercially available, which cleave both upstream and downstream of their recognition sequence at a defined distance (10/12 nucleotides) from this sequence.
This cleavage by the enzyme consequently removes a sequence segment of 2×6+10+12=34 bp. Subsequent religation consequently generates sequences from which 34 base pairs have been removed. Consequently, if a specific cloning has been carried out, the last nucleotide of the endoproteinase recognition sequence and the first nucleotide of the first codon of the protein to be cloned, for example, then lie directly adjacent. Examples of sequences are given in Example 1 in association with a listing of some applications.
Particular advantages of the BcgI system include its tendency to lead in 10-50% of cases, depending on the buffer concentration, to small deletions (3 nucleotides as a rule) at the cleavage site. This then results, in these cases, in the first amino acid of the foreign protein to be expressed, as a rule methionine, no longer being a constituent of the foreign protein, which is to be expressed and which is released, after digestion with the enzyme which recognizes the corresponding protein sequence.
A similar result is obtained when two restriction endonuclease recognition sites are used; i.e. with one of the sites being in, or in the immediate vicinity of, the region of an endoproteinase recognition sequence and the other recognition site, which is ligation-compatible with the former, being located directly upstream of the foreign protein sequence to be expressed such that digestion with the corresponding restriction endonuclease and subsequent religation brings the beginning of the foreign protein sequence to be expressed directly up against the endoproteinase recognition sequence.
REFERENCES:
patent: 5196524 (1993-03-01), Gusatfson et al.
patent: 5434063 (1995-07-01), Lacks
patent: 0161937 (1984-11-01), None
patent: 0293249 (1988-11-01), None
patent: 0 466 332 A2 (1992-01-01), None
patent: 0532043 (1993-03-01), None
patent: 2212160 (1989-07-01), None
Cease, K.B., et al. (1993) Biotech. 14(2), 250-252, 254-255.
Kong, H.K., et al. (1994) J. Biol. Chem. 269(1), 683-690.
Markmeyer, P., et al. (1990) Gene 93, 129-134.
Fish & Richardson P.C. P.A.
November AB Novus Medicatus Berling Gesellschaft fur Molekulare
Patterson Jr. Charles L.
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