Yeast secretory expression of insulin precursors

Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine^B30 as a fusion protein with the S. cerevisiaeα-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine^B29 to glycine^A1, results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic–aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield. Received: 8 February 2000 / Received revision: 2 May 2000 / Accepted: 2 May 2000     

Fermentation and purification of lacZ-fused single chain insulin precursor for (B30-homoserine) human insulin

In order to produce the single chain precursor of a novel human insulin analogue, (B³⁰-homoserine) insulin, the fermentative behaviors ofEscherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused withlacZ gene undertac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was supplemented to fermentation medium after 4 h cultivation ofE. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.     

Secretory expression of insulin precursor in pichia pastoris and simple procedure for producing recombinant human insulin

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.     

Production and purification of single chain human insulin precursors with various fusion peptides

For the production and purification of a single chain human insulin precursor four types of fusion peptides β-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)₆-tagged sequence (HTS) were investigated. RecombinantE. coli harboring hybrid genes was cultivated at 37°C for 1 h, and gene induction occurred when 0.2 mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except forE. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0 mM IPTG, followed by a longer than 4 h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique     

Secretory Expression of Insulin Precursor in Pichia pastoris and 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.     

Expression of synthetic human-lysozyme gene in Saccharomyces cerevisiae: use of a synthetic chicken-lysozyme signal sequence for secretion and processing

A multicopy plasmid was constructed to direct the synthesis and secretion of human lysozyme (HLY) in Saccharomyces cerevisiae. This plasmid contains a synthetic chicken-lysozyme signal sequence (SIG) and a synthetic HLY structural gene, both inserted between the yeast GAL10 promoter and 2 mu plasmid FLP (flip-flop recombination gene) terminator. The resulting plasmid directed the expression of the hybrid pre-lysozyme, with most of the HLY activity secreted into the culture medium and extracellular periplasmic space. The HLY activity in the culture medium increased with cell growth. The yeast accurately processed the hybrid precursor at the junction between the chicken SIG and the coding sequence downstream, yielding mature HLY. HLY purified from the culture medium was homogeneous and displayed specific activity identical to that of authentic HLY.     

Expression of monomeric insulin precursor in Pichia pastoris and its conversion into monomeric B27 Lys destripeptide insulin by tryptic hydrolysis

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition,and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.     

Study on preparation and unique properties of a novel insulin analogue with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain

A novel insulin analog, PIns, with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at human regular insulin B-chain was acquired through gene engineering. Preproinsulin for PIns was cloned and expressed using a bacterial expression system at a high level (72.1%) as fusion protein carrying a modified thioredoxin N-terminal region (1–21) linked to N-terminus of proinsulin by a lysine residue. Purified fusion protein was refolded and converted into PIns by a single enzymatic reaction. After PIns was purified, the homogeneity of it was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectronic focusing electrophoresis, amino acid composition analysis and mass spectrometry methods. A decreased tendency of self-association of PIns as compared with regular insulin was demonstrated by the size exclusion HPLC analysis. When subcutaneously administrated into normal rats, the PIns showed a faster rate of onset of action and a shorter duration of action compared with regular insulin, similar to the pharmacokinetic characteristics of insulin Lispro. These results showed that PIns is a rapid insulin analog. Furthermore, the N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain can be excised by DPPIV and recombinant peptidase with DPPIV-like activities. It is suggested that PIns serves as an artificial insulin precursor and can be transformed to regular insulin in vivo due to the truncation of N-terminal sequence of PIns B-chain by DPPIV.     

A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single-chain des-(b-30)-lnsulin precursor with lysyl endopeptidase

It has been shown that the single-chain des-(B-30)-insulin precursor (SCI) can be converted into human insulin ester by transpeptidation using trypsin in the presence of a threonine derivative. The present study demonstrates that Achromobacter lyticus protease 1 (lysyl endopeptidase) can catalyze the transpeptidation reaction more efficiently than can trypsin. It is also shown that des-(B-30)-insulin (DAI) can be produced by hydrolysis of SCI with the lysyl endopeptidase. Since it is well known that SCI can be produced by gene technology, the following method is recommended for industrial production of human insulin ester: hydrolysis of SCI with lysyl endopeptidase followed by coupling of the resulting DAI with a threonine derivative using trypsin or lysyl endopeptidase.     

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells

The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant human insulin: X simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Expression of Japanese quail ovalbumin inSaccharomyces cerevisiae

A cDNA sequence coding for Japanese quail ovalbumin was used for the construction of expression plasmid under the ADH1 promoter of the yeast shuttle vector pVT101-U. The resulting recombinant expression vector pJK2 was used for the transformation ofSaccharomyces cerevisiae. Expression of quail ovalbumin in yeast cells was demonstrated by Western blotting followed by immunochemical detection.     

In vitro folding/unfolding of insulin/single-chain insulin

Insulin is a double-chain (designated A and B chain respectively) protein hormone containing three disulfides, while insulin is synthesized in vivo as a single-chain precursor and folded well before being released from B-cells. Although the structure and function of insulin have been well characterized, the progress in oxidative folding pathway studies of insulin has been very slow, mainly due to the difficulties brought about by its disulfide-linked double-chain structure. To overcome these difficulties, we recently studied the in vitro oxidative folding process of two single-chain insulins: porcine insulin precursor (PIP) and human proinsulin (HPI). Based on the analysis of the intermediates captured during folding process, the folding pathways have been proposed for PIP and HPI separately. Similarities between the two folding pathways disclose some common principles that govern the insulin folding process. The following unfolding studies of PIP and HPI further indicate that C-peptide might also function during the folding of proinsulin. Here, we gave a brief review on in vitro folding/unfolding process of insulin and single-chain insulin. The implication of these studies on protein folding has also been discussed.     

Construction of a family of artificial genes of human insulin

Using oligonucleotide-directed mutagenesis and chemical-enzymatic DNA synthesis, genes for A and B insulin chains, C-peptide and Tyr-C-peptide have been constructed starting from synthetic gene for human proinsulin synthesized earlier. The genes for human preproinsulin, mini-proinsulin, single-chain insulin and their modifications were also synthesized. The constructions obtained were cloned in plasmid vectors.     

猪胰岛素前体在酵母Kluyveromyces lactis中的分泌表达及其转化为人胰岛素

通过将包括猪胰岛素前体（ＰＩＰ）基因在内的表达框架克隆至质粒ｐＫＤ１衍生的两种载体上而在酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓｌａｃｔｉｓ中分泌表达猪胰岛素前体。根据放射免疫测定结果,猪胰岛素前体的表达水平为２０～３０ｍｇ／Ｌ。猪胰岛素前体经过转肽被转变为基因工程人胰岛素。分析结果表明,来自Ｋ．ｌａｃｔｉｓ的人胰岛素,其氨基酸组成、晶体形状和生物活力与天然胰岛素相同。     

文昌鱼胰岛素样肽的表达、纯化、鉴定及其多克隆抗体制备

为了深入研究胰岛素和胰岛素样生长因子1（IGF－1）的起源和进化以及结构与功能的关系。表达了胰岛素和IGF－1的祖先分子－－文昌鱼胰岛素样肽（ILP）。重组单链ILP的基因用化学方法合成（从cDNA推测的ILPB结构域的C端和A结构域的N端用Ala-Ala-Lys三肽连接起来,并钭B28Arg突变为Lys）,克隆到表达载体pVT102-U中,ILP在酿酒酵母中得到有效表达。发酵液经4步分离纯化,得到均一的单链ILP,经质谱测定分子量和氨基酸组成分析证明表达产物正确。通过Lys-C蛋白内切酶处理将重组单链ILP转化成双链形式。虽然双链ILP与人胰岛素受体没有结合活力。但圆二色性光谱显示它与胰岛素的结构非常相似,用表达的单链ILP免疫新西兰大白兔,获得了高滴度的多克隆抗体。     

The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling

A cloned approximately 5 kb cDNA (human placenta) contains the coding sequences for the insulin receptor. The nucleotide sequence predicts a 1382 amino acid precursor. The alpha subunit comprises the N-terminal portion of the precursor and contains a striking cysteine-rich "cross-linking" domain. The beta-subunit (the C-terminal portion of the precursor) contains a transmembrane domain and, in the intracellular region, the elements of a tyrosine phosphokinase: an ATP-binding site and a possible tyrosine autophosphorylation site or sites. The overall structure is reminiscent of the EGF receptor; the cross-linking domain of the alpha subunit and several regions of the beta subunit exhibit sequence homology with the EGF receptor. The phosphokinase domain also exhibits homology with some oncogenic proteins that have tyrosine phosphokinase activity, in particular, a striking homology with v-ros. Southern blotting experiments suggest that the coding region spans more than 45 kb. The insulin receptor gene is located on chromosome 19.     

一种新的B链C端去四肽胰岛素的研制方法

报道了将单体胰岛素前体(MIP)经胰蛋白酶和羧肽酶B两步连续酶切获得B链C端去四肽胰岛素(DTI)的方法。MIP由甲醇酵母表达,最高发酵表达量达到150mg/L。发酵液中MIP通过疏水层析,分子筛初步纯化后直接进行酶切,在胰蛋白酶酶切3h后加入抑制剂paminobenzamidine处理15min,然后直接加入羧肽酶B酶切6h,再通过反相柱纯化即可得到纯品DTI,从分子筛到最后DTI,总纯化得率达到77%。按中国药典小白鼠惊厥法测定得DTI的生物活力为22IU/mg,是胰岛素的80%,在Superdex G-75分子筛上测定DTI的解离聚合曲线,证明其是单体。     

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Key words proinsulin; C-peptide; Pichia pastoris