Process for the enzymatic cleavage of recombinant proteins using

Details Process for the enzymatic cleavage of recombinant proteins using Process for the enzymatic cleavage of recombinant proteins using

1992-08-30

1995-06-27

Patterson, Jr., Charles L.

Chemistry: molecular biology and microbiology

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

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

435 6952, 4352523, 43525233, 4353201, 530351, 536 234, C12P 2102, C12N 1524, C07K 14535

Patent

active

054279275

DESCRIPTION:

BRIEF SUMMARY
The invention concerns a process for the sequence-specific cleavage of proteins obtained biotechnologically using IgA proteases (also denoted Igases) and in particular a process for the production of recombinant proteins or peptides in prokaryotes and the subsequent removal of a N-terminal sequence.
The biotechnological preparation of proteins is preferably carried out using microorganisms which can be easily cultured and which allow the isolation of the protein which is produced in a simple manner. Suitable microorganisms for this are e.g. the gram-negative bacterium Escherichia coli, the gram-positive bacterium Streptococcus carnosus as well as the baker's yeast Saccharomyces cerevisiae. The expression of authentic foreign genes in such microorganisms is, however, often disadvantageous. In E. coli, for example, the amino-terminal methionine residue of natural proteins which is a result of translation is usually efficiently cleaved off by proteases. In the case of foreign proteins the first methionine residue is, however, usually only partially cleaved off. A suitable procedure for the production of such proteins with a defined amino end is therefore first to produce these in the form of fusion proteins and subsequently to cleave them in a defined way with a sequence-specific protease.
Compared to authentic proteins, such fusion proteins can in addition have the advantage that they aggregate in the cell of the microorganism and form dense precipitation bodies ("inclusion bodies") which can easily be separated from other cellular components and thus facilitate the isolation and purification of the desired protein. On the other hand, carrier proteins which are initially fused to the actual desired protein by means of genetic engineering procedures impart a particular stability against unspecific proteolytic degradation to the fusion partner; the problem of degradation of polypeptides which are recognized as being foreign concerns in particular the biotechnological preparation of small peptides. Furthermore, other carrier proteins allow the desired proteins to be directed into particular cell compartments from which they can be particularly easily purified and where they are particularly stable and/or where they are accessible for test purposes. Finally, carrier proteins can also have special properties which allow an efficient purification e.g. by affinity chromatography. For most application purposes fusion proteins are preferred which carry the carrier protein at the amino end and the desired protein at the carboxyl end. However, the opposite version or the coupling of the desired protein with two fusion partners can also be desirable in particular cases. In addition, the reiteration of the desired protein within one fusion can be advantageous.
In order to obtain the desired protein in a free form from such a fusion, it is necessary to cleave the covalently bound fusion partners from one another. In principle this can be achieved by chemical or biochemical (enzymatic) methods. However, the limited specificity of the methods which have been hitherto available is a limitation in this process, for in order to obtain the desired protein it is important that such a cleavage takes place in a cleavage sequence between the fusion partners, i.e. the junction region, but under no circumstances additionally within the desired protein itself. The cleavage of the fusion partners must therefore be carried out highly specifically.
Chemical processes which have been used up to now for the sequence-specific separation of fusion proteins are for example the cleavage by cyanogen bromide at the amino acid methionine within a protein and the cleavage between the amino acids Asp.!.Pro in an acid medium using formic acid. These processes are only then suitable when the specific cleavage site in the desired protein does not occur again apart from in the region of the junction to the fusion partner. However, in general, biochemical cleavage procedures are preferable to chemical methods because the former can usually be carried ou

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