Enzymatic process for producing a desired protein from an amino

Details Enzymatic process for producing a desired protein from an amino Enzymatic process for producing a desired protein from an amino

1997-02-05

1998-07-21

Weber, Jon P.

Chemistry: molecular biology and microbiology

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

Enzymatic production of a protein or polypeptide

C12P 2106

Patent

active

057834130

DESCRIPTION:

BRIEF SUMMARY
The invention relates to an enzymatic process for producing a desired protein from an amino terminal extended protein.
Over the past decade, the principle of a new protein purification technique has emerged as a result of recombinant DNA technology. DNA, encoding additional polypeptide or protein tags, is fused to the gene of interest. Expression of these gene fusions results in protein fusions which may be identified, analyzed and purified by techniques using the properties of the additional polypeptide tag. This has in certain cases eliminated the need for extensive screening and optimization procedures previously required for purification.
Polypeptide tags specifying a variety of different biospecific and biochemical interactions can be or have been utilized as the basis for fusion tag techniques. These include (1) entire enzymes with affinity for substrates or inhibitors; (2) peptide-binding proteins; (3) carbohydrate-binding proteins or domains; (4) biotin-binding domains; (5) antigenic epitopes with affinity for specific immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) polyhistidine residues for binding to metal chelates and subsequent recovery by immobilized metal affinity chromatography and (8) other polyamino acids with various binding specificities.
Provided that the added polypeptide tag can be effectively and specifically removed, this technology should find application in areas from basic research to industrial production. On a laboratory scale, the fusion tag technique should be a powerful and elegant tool for one-step recovery and purification of a large number of authentic recombinant proteins. On an industrial scale, the technology could be used in the recovery and purification of high-cost pharmaceuticals and other proteins prepared by using recombinant technology or other technology.
Unfortunately, the use of this protein purification principle has been very limited because of a major and serious drawback. It is often difficult, impossible or too expensive to remove the additional terminal sequence from the desired protein product. This is most often necessary to do in order to obtain the desired correct protein function, structure and other properties.
This is of the utmost importance when dealing with basic research covering e.g. function studies of proteins (enzymes, receptors, hormones, etc.).
It is also of the utmost importance when dealing with proteins for pharmaceutical use in which case it is essential to avoid undesired immunological and other response effects of the pharmaceutically active protein which therefore has to have the correct and natural structure, i.e. without any added polypeptide or single amino acid.
One solution to the problem has been to remove the added polypeptide tag by using relatively drastic conditions and chemical cleavage reagent, such as CNBr in 70% formic acid, or hydroxylamine treatment at basic pH. The methods have several major drawbacks which include the use of protein-destroying conditions and very toxic chemicals, this leading to high levels of unwanted degradation of the protein of interest and serious health problems for the personnel involved in the use of these techniques.
Another approach is to use so-called specific endoproteases. The blood clotting factor Xa has a proteolytic specificity for the tetra peptide sequence IleGluGlyArg and has been used to liberate different added sequences from different proteins. A sequence allowing a relatively specific cleavage by collagenase has also been proposed. Another sequence of five amino acids can be cleaved by the enzyme enterokinase.
Another example using this technique is known from DE 29 22 496 which partly corresponds to U.S. Pat. No. 4,543,329. This document discloses the preparation of proteins from C-terminal extended proteins. The protein is extended with 4 amino acids, where the extension is Pro-Xyz-Gly-Pro, and Xyz is any amino acid. The tetrapeptide is removed using the enzymes collagenase, an aminopeptidase and a proline aminope

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