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Purification of an L ‐asparaginase—atrial natriuretic peptide fusion protein expressed in Escherichia coli
Author(s) -
Ma NienTung,
Wang YunFang,
Roe Bruce A.,
Harrison Roger G.
Publication year - 1995
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260470410
Subject(s) - fusion protein , escherichia coli , chemistry , affinity chromatography , signal peptide , peptide , biochemistry , isoelectric point , molecular mass , peptide sequence , microbiology and biotechnology , biology , gene , recombinant dna , enzyme
Abstract A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L ‐Asparaginase from Erwinia chrysanthemi is fused to the N‐terminus of a model peptide, α‐human atrial natriuretic peptide (α‐hANP). L ‐Asparaginase was chosen because of its selective affinity for L‐asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L ‐asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L ‐asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of α‐hANP was observed by coupled HPLC/mass spectrometry. © 1995 John Wiley & Sons Inc.