Enhanced proteolytic cleavage of recombinant fusion proteins

Details Enhanced proteolytic cleavage of recombinant fusion proteins Enhanced proteolytic cleavage of recombinant fusion proteins

1994-07-12

1995-05-16

Wax, Robert A.

Chemistry: molecular biology and microbiology

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

Enzymatic production of a protein or polypeptide

435 697, 435176, 435177, 435178, 435179, 435180, 435181, 530407, 935 47, 935 51, C12P 2106, C12N 1112, C12N 1106, C12N 1108

Patent

active

054160073

ABSTRACT:
Disclosed are methods for increasing the yield of intact target proteins by cleaving fused polypeptides made by recombinant DNA techniques. The fused polypeptides are designed at the DNA level to have a preselected primary cleavage site in a pendant polypeptide fused to a protein of interest. Structural features of the fused polypeptide and cleavage reaction environment are controlled to favor cleavage by a preselected cleavage agent at the primary cleavage site over a second cleavage agent-sensitive amino acid sequence in the target protein. The cleavage reaction is terminated before completion when the ratio of intact target protein to truncated, cleaved target protein is optimized, and the remaining reaction mixture comprising uncleaved fused polypeptide is resubjected to the cleavage agent. The presence of charged organic molecules in the cleavage reaction mixture favors cleavage at the primary cleavage site. The endopeptidase used for cleavage may be immobilized on an insoluble support matrix.

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