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.

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

“…In agreement with previous mutagenesis studies of BPTI (25)(26)(27)(28), the replacement of the large basic Lys residue against the smaller hydrophobic leucine residue transformed rShPI-1/K13L into a high-affinity PPE inhibitor. This additional activity has never been observed for natural or wild-type rShPI-1A, even if an I o /E o molar ratio of 170 is used (21,37). The PPE inhibition activity was stable after incubation for 15 s, indicating that the enzyme, the inhibitor, and the substrate as well as their complexes were in equilibrium.…”

“…Cloning and Production of ShPI-1/K13L-The gene of ShPI-1A was amplified by PCR with site-specific primers using the plasmid pBM301 as a template; it was obtained previously for rShPI-1A expression (37). The purified product was cloned into the XhoI/XbaI-digested pPICZ␣A vector (Invitrogen).…”

“…These primers carried the desired mutations (underlined) and an XmaJI (AvrII) restriction site (C2CTAG) for detection of positive clones. The variant protein was produced, purified, and stored following the previously established protocol for recombinant wild-type rShPI-1A (37). Protein production and purification was followed by SDS-PAGE (39).…”

“…Inhibitory Activity of rShPI-1/K13L-Incubation of trypsin, chymotrypsin, and HNE with rShPI-1/K13L resulted in an inhibition of these serine proteases consistently characterized by K i values within the nanomolar range (Table 2) as described previously for the natural and recombinant wild type-like inhibitors (21,37). Solely the binding affinity to trypsin is significantly decreased (ϳ100-fold) as a result of the mutation, whereas rShPI-1/K13L appears to be slightly more potent against chymotrypsin and HNE than the wild-type inhibitor.…”

“…This molecule inhibits not only serine proteases but also cysteine and aspartic proteases such as papain and pepsin with K i values in the nanomolar range (21), qualifying ShPI-1 for biotechnological use (37). We recently presented the three-dimensional structure of free and trypsin-bound recombinant ShPI-1 (rShPI-1A) (35,38), revealing a high degree of conservation for the intermolecular interactions around the basic P1 residue (Lys 13 ) compared with homologous complexes of mammalian inhibitors.…”