title: Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris.
abstract: Pichia 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-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris.
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Plaza de la Revolución, La Habana, Cuba.
authors.forename: Dayrom F
authors.initials: DF
authors.lastname: Gil
authors.forename: Rossana
authors.initials: R
authors.lastname: García-Fernández
authors.forename: Maday
authors.initials: M
authors.lastname: Alonso-del-Rivero
authors.forename: Emilio
authors.initials: E
authors.lastname: Lamazares
authors.forename: Mariela
authors.initials: M
authors.lastname: Pérez
authors.forename: Laura
authors.initials: L
authors.lastname: Varas
authors.forename: Joaquín
authors.initials: J
authors.lastname: Díaz
authors.forename: María A
authors.initials: MA
authors.lastname: Chávez
authors.forename: Yamile
authors.initials: Y
authors.lastname: González-González
authors.forename: Manuel
authors.initials: M
authors.lastname: Mansur
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000096:biosynthesis* / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D015870:Gene Expression; D006801:Humans; D060847:Metabolic Engineering; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012615:Sea Anemones / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C098973:recombinant Kunitz protease inhibitor; C060793:miniproinsulin; D011384:Proinsulin; D007611:Aprotinin