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Characterization of the proteases involved in the N‐terminal clipping of glucagon‐like‐peptide‐1‐antibody fusion proteins
Author(s) -
Dorai Haimanti,
Santiago Aleixo,
Campbell Marguerite,
Tang Q. Mike,
Lewis Michael J.,
Wang Yonghui,
Lu QiaoZhen,
Wu ShiawLin,
Hancock William
Publication year - 2011
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.537
Subject(s) - proteases , protease , biochemistry , exopeptidase , peptide , fusion protein , protein degradation , serine , proteolysis , biology , amino acid , chemistry , enzyme , recombinant dna , gene
In an attempt to develop high producing mammalian cell lines expressing glucagon‐like‐peptide‐1‐antibody fusion proteins (GLP‐1), we have noted that the N‐terminal GLP‐1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N‐terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine‐threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011