Open AccessRecombinant expression of S h PI ‐1 A , a non‐specific BPTI ‐ K unitz‐type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in P ichia pastoris
Author(s) -
Gil Dayrom F.,
GarcíaFernández Rossana,
AlonsodelRivero Maday,
Lamazares Emilio,
Pérez Mariela,
Varas Laura,
Díaz Joaquín,
Chávez María A.,
GonzálezGonzález Yamile,
Mansur Manuel
Publication year - 2011
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2011.00749.x
Subject(s) - pichia pastoris , biology , proteases , recombinant dna , proteolysis , biochemistry , protease inhibitor (pharmacology) , heterologous expression , protease , serine protease , serine , protein degradation , microbiology and biotechnology , enzyme , gene , virology , human immunodeficiency virus (hiv) , antiretroviral therapy , viral load
P ichia pastoris is a highly successful system for the large‐scale expression of heterologous proteins, with the added capability of performing most eukaryotic post‐translational modifications. However, this system has one significant disadvantage – frequent proteolytic degradation by P . pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI ‐ K unitz protease inhibitor S h PI ‐1 isolated from the sea anemone S tichodactyla helianthus , was expressed in P . pastoris . The recombinant inhibitor (r S h PI ‐1 A ), containing four additional amino acids ( EAEA ) at the N ‐terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. r S h PI ‐1 A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. r S h PI ‐1 A protected a model protein, recombinant human miniproinsulin (rh MPI ), from proteolytic degradation during expression in P . pastoris . The addition of purified r S h PI ‐1 A at the beginning of the induction phase significantly protected rh MPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as r S h PI ‐1 A can be used to improve the yield of recombinant proteins secreted from P . pastoris .