Secretory Expression of Insulin Precursor inPichia pastorisand Simple Procedure for Producing Recombinant Human Insulin

Abstract

In this work, Pichia pastoris was applied to produce human insulin by a simple procedure. The synthesized insulin precursor (ILP) gene was inserted into pPIC9K to obtain secretary expression plasmid pPIC9K=ILP. Pichia pastoris GS115 was transformed by pPIC9K=ILP and the high expresser was screened. In a 16 L fermentor, the insulin precursor production was 3.6 g=L. Insulin precursor, purified by one-step chromatography, was converted into human insulin by transpeptidation. The yield of the processing procedure from insulin precursor to insulin reached up to 70%. In vivo assay showed that the biological activity of the produced recombinant human insulin was 28.8 U=mg.

A Simplified and Efficient Process for Insulin Production in Pichia pastoris

“…This high-level secretory production of IP in the culture supernatant compares favorably to the highest yields previously published of 1.5 to 3.84 g L -1 [6,7,11] and is significantly higher than others published which are in the range of 0.18 to 0.3 g L -1 [12–15]. …”

Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

“…Secretion yields of the single chain insulin precursors can be enhanced by adding a charged synthetic spacer peptide in front of the insulin B-chain segment, which allows more efficient Kex2 endoprotease processing after the α-factor pre-pro leader [13,22,32]. After recovery from the culture broth, IP can be converted enzymatically into human insulin [26,27,29,33,34]. …”

Two‐step transpeptidation of the insulin precursor expressed in Pichia pastoris to insulin ester via trypsin‐catalyzed cleavage and coupling

“…P. pastoris expressing the recombinant human insulin precursor fusion protein (EEAEAEAEPK‐B 1–29 ‐AAK‐A 1–21 ) was constructed and stored in the laboratory .…”

“…However, the expression of a cDNA encoding a proinsulin‐like molecule with the deletion of threonine B30 , followed either by the replacement of the human proinsulin C‐peptide with a small C‐peptide (RR, AAK, or EWK) or by direct fusion of lysine B29 to glycine A1 and the subsequent fusion to the α‐factor leader, resulted in the efficient expression of single‐chain proinsulin‐like molecules . Using this strategy, a recombinant P. pastoris strain that could efficiently express the single‐chain insulin precursor, consisting of the first 29 amino acids of the human insulin B chain ( B1 – B29 ), linked to 21 amino acids of the human insulin A chain ( A1 – A21 ) via the mini C‐peptide AAK connecting to lysine B29 and glycine A21 was constructed in our laboratory . In the strain, a spacer peptide (EEAEAEAEPK) localized between the leader peptide (signal peptide) and insulin precursor was inserted to improve Kex2 endoprotease processing and insulin precursor fermentation yield.…”

“…The insulin precursor fusion protein was transformed into human insulin ester via one‐step enzymatic transpeptidation in the presence of high concentrations of trypsin and H‐Thr(tBu)‐OtBu to ensure a high conversion ratio . One‐step transpeptidation consists of two reactions converting insulin precursor fusion protein into human insulin ester.…”