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High‐level production and isolation of human recombinant α 1 ‐proteinase inhibitor in yeast
Author(s) -
Hoylaerts M.,
Weyens A.,
Bollen A.,
Harford N.,
Cabezón T.
Publication year - 1986
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(86)81391-6
Subject(s) - agarose , recombinant dna , affinity chromatography , microbiology and biotechnology , yeast , complementary dna , plasmid , polyethylene glycol , biochemistry , biology , sepharose , chemistry , enzyme , gene
The cDNA coding for mature human α 1 ‐proteinase inhibitor (α 1 ‐PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated α 1 ‐PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2μ and the leu2D selection marker. Biologically active recombinant α 1 ‐PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high‐ M r , yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE‐agarose, Zn‐chelate agarose, κ‐chain agarose, heparin‐agarose and aminohexyl‐agarose.