Process for the production of peptides by way of streptavidin fu

Details Process for the production of peptides by way of streptavidin fu Process for the production of peptides by way of streptavidin fu

1998-06-25

2000-10-24

Carson, Karen Cochrane

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

536 234, 530399, 530402, 4353201, 4352523, 4352542, C12N 1509, C12N 120, C97H 2104, A61K 3824, C07K 1400

Patent

active

061365645

DESCRIPTION:

BRIEF SUMMARY
The invention concerns a process for the recombinant production of peptides by expression of fusion proteins with streptavidin and subsequent enzymatic cleavage of the fusion protein.
Peptides are usually understood as substances which are composed of up to ca. 100 amino acids. Such peptides can either be prepared chemically (Kent, S. B. H. et al. (1988) (1), Hodson, J. H., (1993) (2) or recombinantly (Kopetzki, E. et al. (1994) (3) Winnacker, E. -L. (1987) (4), Harris, T. J. R. (1983) (5)).
The disadvantages of chemical peptide synthesis are in particular that an economic synthesis is only possible up to ca. 30 to 40 amino acids and undesired modifications (false sequences, non-cleaved protective groups) are frequently formed during the synthesis. Further problems are racemization during fragment coupling, difficulties in cleaving the protective groups and finally the complicated purification.
Various methods can be used for the recombinant production of peptides. For example a direct expression can take place in the cytoplasm of microorganisms or cell lines. However, a minimum polypeptide length of ca. 80-100 amino acids is required for this. Smaller peptides are not stable and are degraded by proteolysis. Moreover these proteins usually contain an additional N-terminal methionine and the yields are very low.
The production of such peptides can be improved by the expression of soluble fusion proteins with a selective cleavage sequence and subsequent release of the desired peptide by chemical or enzymatic cleavage (Itakura, K. et al. (1977) (20); EP-B 0 001 930 (21); Gram, H. et al. (1994) (19); Sharma, A. et al., (1992) (22)). However, a particular disadvantage of soluble fusion proteins is that they can already be degraded by proteolysis in the cell or during secretion and processing mainly in the non-structured peptide region.
The production of streptavidin fusion proteins is described in EP-B 0 198 015, by Sano, T. (1991) (9) and Sano, T. (1992) (10). Such chimeric proteins in the case of Sano comprise the amino acids 16-133 of streptavidin as the streptavidin moiety, a polylinker and the sequence of the target protein. The target proteins described by Sano are the mouse metallothionein I protein and the T7 gene 10 protein. However, these chimeric proteins contained no cleavage site by means of which the target protein can be cleaved again from the streptavidin moiety. The production of streptavidin fusion proteins in Streptomyces is described in EP-B 0 198 015. However, the isolation of the peptide component from the fusion protein is very complicated. Thus for example an affinity chromatography using iminobiotin as the ligand must be carried out both before and after the cleavage.
The object of the present invention was to provide a process by which peptides can be produced via streptavidin fusion proteins in a high yield and purity with separation from the streptavidin component that is as complete as possible.
The object is achieved according to the invention by a process for the recombinant production of a peptide by expression of a DNA in microorganisms, preferably prokaryotes, which codes for a fusion protein comprising streptavidin and the said peptide wherein streptavidin and the peptide are linked via a peptide sequence which can be cleaved by an endoproteinase, isolation of the insoluble inactive fusion protein, solubilization of the inactive protein with a denaturing agent, dilution of the denaturing agent at a pH value between 8.5 and 11 until it is possible to cleave the fusion protein by an endoproteinase, cleavage of the fusion protein, lowering the pH value until the cleaved streptavidin and non-cleaved fusion protein precipitate and purification of the desired peptide from the supernatant.
The process according to the invention utilizes the advantage that streptavidin fusion proteins can be expressed very well in prokaryotes and can be isolated in the form of insoluble inactive proteins (inclusion bodies). Streptavidin fusion proteins solubilized in denaturing agents can be diluted a

REFERENCES:
Saito et al. Journal of Biochemistry, vol. 102, No. 1, Jul. 1987, pp. 111-122, "Bacterial synthesis of recombinant alpha human atrial natriuretic polypeptide".
Gardella et al. Journal of Biochemistry, vol. 265, No. 26, Sep. 15, 1990, pp. 15854-15859, "Expression of human parathyroid hormain-(1-84) in Escherichia coli as a Factor X-cleavable fusion protein".
Wingender et al., Journal of Biochemistry, vol. 264, No. 8, Mar. 15, 1989, pp. 4367-4373, "Expression of human parathyroid hormone in Escherichia coli".

Affiliated with

Also associated with

Rating

Process for the production of peptides by way of streptavidin fu does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Process for the production of peptides by way of streptavidin fu, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for the production of peptides by way of streptavidin fu will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-1962292