HUT50494A - Process for producing peptide analogs in large numbers, as well as new type peptide analogs - Google Patents

Abstract

A method of generating a large number of analogs of a selected peptide will be described. The method comprises the following steps: synthesizing two different mixtures of identically long oligonucleotides having different bases at some specific positions and some identical bases at the 3-end, mixing the two mixtures to form a mixture of partially double-stranded, equally long DNA sequences, which are then enzymatically converted into completely double-stranded DNA sequences, cutting the latter mixture with two different enzymes to produce DNA sequences that can be ligated to a similarly cut vector, inserting the DNA sequences into the vector by ligation to generate a mixture of vectors, which are then transformed into a host in a manner known per se, proliferating the host to form colonies which are analyzed sequentially to detect the DNA sequences encoding a single protein encode, under expression conditions separate growth of the large number of hosts to produce a large number of analogs of the chosen peptide. In addition, new analogs of VIP and a plasmid containing a gene encoding one of the novel analogs of VIP are disclosed. manufacture; Peptide; oligonucleotides; bases; 3 'end; DNA sequences; enzymes; Vector}

Description

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EXTRACT

A method for producing a large number of peptide analogs and novel type peptide analogs

Szeged Biological Center of HAS, SZEGED

Date of announcement: 1988 12. 22.

Topic: 23. 12. 1987 (8705139-7)

SWEDEN

The present invention relates to a method for producing a large number of analogs of a particular peptide. They act like that

- having the same length as the chemical synthesis, in a few specific locations, and two different mixtures of oligonucleotides containing the same nucleotide at the 3 'end;

mixing said two oligonucleotide mixtures into a partially double-stranded mixture of DNA molecules of the same length, which are then converted completely into double-stranded DNA molecules after enzymatic sequencing;

- the double strand. A mixture of DNA molecules is cleaved by two different enzymes to produce DNA molecules ligated with a similarly cleaved DNA vector;

by incorporating DNA molecules into the DNA vector by ligation, a mixture of DNA vectors is then transformed with DNA vectors, in a manner known per se;

- transformants are cultured and the nucleotide sequence of the DNA molecules encoding each protein is determined by examining the colonies thus obtained;

- a large number of analogues are produced by the large number of analogs of the particular peptide, under high conditions for expression in a plurality of transformants.

The invention also relates to a method for producing a plasmid DNA carrying a gene encoding one of the novel vasoactive intestinal polypeptide analogs and one of the novel vasoactive intestinal polypeptide analogs.

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A method for producing a large number of peptide analogs and novel type peptide analogs

Szeged Biological Center of HAS, SZEGED

inventors:

Tamás BÁRTFAI, STOCKSUND, SWEDEN,

TJÖRNHAMMAR Marie-Louise, STOCKHOLM, SWEDEN,

András SIMONCSITS, STOCKHOLM, SWEDEN,

Miklós KÁLMÁN, SZEGED,

Imre CSERPÁN, SZEGED

Date of announcement: 1988 12. 22.

priority:

1987-12-23.

(8705139-7)

SWEDEN

The present invention relates to a method for producing a large number of peptide analogues and novel type peptide analogs.

The present invention relates primarily to a process for the production of plasmid DNAs comprising a large number of analogues or novel type analogs of the vasoactive intestinal polypeptide (VIP), as well as genes comprising DNA sequences encoding the vasoactive intestinal polypeptide analogs of the invention.

The pharmaceutical industry, as well as independent researchers, are constantly testing novel compounds that are slightly different from known useful compounds to be more effective, specific, and so on. derivatives. When said known compound is a peptide or polypeptide, the amino acid sequence of this compound may be replaced by one or more amino acids to other natural or non-natural amino acids.

It would be desirable from the point of view of the active screening assays that several analogs of the selected peptide can be produced simultaneously, saving both money and time.

• *

The method of the present invention allows the simultaneous production of a number of analogues of a selected protein with only L-amino acids. As described herein, analogs of the vasoactive intestinal polypeptide are prepared.

The vasoactive intestinal polypeptide (VIP) is a highly basic peptide comprising a 28 amino acid residue at the C-terminal end that encodes the glucagon secretin protein family. The vasoactive intestinal polypeptide was first distinguished from S. Said and V. Mutt from tissues from the upper intestinal tract in 1970 [Said, SI; Mutt, V., Science, 169, 1217-1218 (1970)], but later demonstrated in nerve tissue and internal separation in glandular cells [Said, SI, Vasoactive Intestinal Peptide, Raven Press, NY (1982), and Said, SI. Peptides, pp. 143-150 (1984)]. The biological effects of vasoactive intestinal polypeptide include expansion of cerebral blood vessels, stimulation of prolactin release, stimulation of pancreatic exocrine secretion, effect on penile erection [Said, SI, Vasoactive Intestinal Peptide, Raven Press, NY (1982); Rosténe, WH, Progress in Neurobiology, 22, 103-129 (1984); Mutt, V., Brain Peptides, eds. Krieger, DT et al. (1983)], and the enhancement of cholinergically stimulated salivation [Lundberg, JM, et al.

Acta Physiol. Scand., 114, 329-337 (1982)]; the vasoactive intestinal polypeptide further participates in the formation of the surfactant fluid film in the lung [Barnes, PJ, TIPS, 8, 24-27 (1987)].

The precursor gene encoding the human vasoactive intestinal polypeptide has recently been isolated and the nucleotide sequence has been determined [Bodner, M. et al., Proc. Natl. Acad. Sci., USA, 82, 3548-3551 (1985); Lindner, S. et al., Proc. Natl. Acad. Sci., USA, 84, 605-609 (1987)].

According to the nucleotide sequence data, additional triplets are found at the 3 'end of the gene encoding the vasoactive intestinal polypeptide: these encode a glycine lysine arginine tripeptide. It can be shown that glycine-lysine arginine tripeptide on the C-terminus, or glycine alone, can be a substrate for the peptide-glycidin-a-aridating monooxygenase enzyme (PAMase), which is found to catalyze C-terminal amidation in various tissues [Bradbury, AF. et al., Natré, 298, 6δ6-688 (1982); Eipper, BA et al., Peptides, 4, 921-928 (1983); Glembiski, GC et al., J. Bioi. Chem., 259, 6385-6392 (1984); Gomez, S. et al., FEBS Lett., 167, 160-164 (1984); Eipper, BA et al., Endocrinology,

116: 2497-2504 (1985) and Ouafik, H. et al., Proc. Natl. Acad. Sci., USA 84: 261-264 (1987)].

Obviously, it would be advantageous to produce large amounts of peptide hormones with a wide range of biological effects, the vasoactive intestinal polypeptide using genetic engineering techniques. Although the precursor gene of the human vasoactive intestinal polypeptide has already been isolated and characterized (Bodner, M. et al., Proc. Natl. Acad. Sci., USA, 82, 3548-3551 (1985); LincLer, S. et al., Proc. Natl. Acad. Sci., USA, 84, 605-609 (1987)], the expression of the gene in another organism has not been published so far.

The vasoactive intestinal polypeptide analogs of the invention are produced in E. Coli cells.

The present invention relates to a method for producing a large number of analogs of a particular peptide, comprising:

- preparing a mixture of oligonucleotides of the same length as the chemical synthesis so that the oligonucleotides are different at some specific sites and carry the same base on the 3 'end;

mixing the above oligonucleotide mixture with another similarly prepared oligonucleotide mixture so that some bases of the oligonucleotides of the first mixture at the 3 'end are complementary to some bases of the oligonucleotides of the other mixture at the 3'end; a mixture of partially double-stranded DNA segments of the same length obtained after the coupling of complementary bases is then made into a mixture of fully double-stranded DNA molecules after enzymatic sequencing;

- the DNA molecules encoding the cleavage site of a restriction enzyme endonuclease at the 3 'end of the double stranded endonuclease, and a restriction site of another restriction endonuclease enzyme at their 5' end, are cleaved simultaneously or sequentially with the two enzymes cleaved in the same manner. '3' and 5'-terminated DNA molecules ligated to the end;

- DNA molecules cleaved by the above procedure were ligated into the DNA vectors cleaved as above, and thus obtained

Claims (1)

DNA vector mixture is transformed into DNA host cells in a manner known per se; - culturing the transformant cells and assaying the colonies thus obtained to determine the nucleotide sequences of the DNA molecules: they encode a protein; - A large number of transformants carrying DNA vectors containing the identified protein coding sequences are cultured separately under appropriate conditions for expression, with a high number of peptides. analog. Examples of DNA host cells useful in carrying out the above described process of the invention include, for example, bacteria such as Bacillus species or Escherichia coli; and yeasts such as Saccharomyces cerevisiae. Examples of DNA vectors used in the process include bacteriophages and plasmids. where Threonine X, according to, proline or alanine; Y threonine, lysine, isoleucine or alanine; Z threonine, lysine, isoleucine or arginine; and W denotes an amino or carboxyl group or glycine or glycine lysine arginine tripeptide (these analogs are obtained according to the method of the invention: these analogs were originally intended to be produced); or the amino acid sequence of said vasoactive intestinal polypeptide analogs with the above amino acid sequence is substantially the same, but: - replacing histidine at position 1 with aspartic acid if it is X = alanine, Y = isoleucine and Z = threonine; - replacing histidine at position 1 with tyrosine if X = proline, Y = threonine and Z = lysine; - substituting tyrosine at position 2 and aspartic acid at position 8 with tyrosine if X = threonine, Y = threonine and Z = threonine;