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     

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     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

Expression,Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ^?) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of ～17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays ～50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.     

Isolation of a catabolic product of 2-butynedioic acid that acts as a precursor of nicotinamide adenine dinucleotide

The de novo nicotinamide adenine dinucleotide biosynthetic pathway ofEscherichia coli was investigated; using a cell-free extract of anadB mutant, we synthesized a precursor of quinolinic acid, a key intermediate in the pathway, from¹⁴C-aspartic acid. The synthesis of this compound was repressible by nicotinic acid. This compund was partially purified by ion-exchange column chromatography and then further purified and characterized by ascending partition chromatography on thin-layer plates. A metabolic scheme is presented which hypothesizes that the isolated precursor of quinolinic acid results from the amination of 2-butynedioic acid.     

Secretory expression of α single-chain insulin precursor in yeast and its conversion into human insulin

A synthetic single-chain porcine insulin precursor (PIP) gene and an α-mating factor leader sequence (αMFL) gene obtained by the PCR method are inserted between the promoter and 3'-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/α MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cererisiae transformed by pVT102-U/αMFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.     

An efficient trypsin digestion strategy for improving desB30 productivity from recombinant human insulin precursor fusion protein

The insulin precursor (IP) expressed in Pichia pastoris is a single-chain peptide fused with a spacer peptide (EEAEAEAEPK) localized at its N-terminus and containing three trypsin cleavage sites in the polypeptide chain. The IP fusion protein is trypsinized to generate the insulin product desB30, which has a deletion of threonine^B30. The three restriction sites on IP fusion protein had different affinities for trypsin and were digested in sequential order. Further analysis showed that approximately 20% of the IP digestion intermediates could not be converted into the final desB30 product if the IP fusion protein was digested in an aqueous phase. This result can be attributed to the formation of IP dimers or hexamers, which could restrict enzyme reactivity in the aqueous phase. To enhance the conversion yield of the IP fusion protein to desB30 products, a new digestion method was established. The IP was digested in the eluent that resulted from reverse phase chromatography during the purification process, which improved the yield of digestion from 80.2% to 95.6%.     

Characterization of skeletal muscle insulin receptor

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.     

A rat kidney neutral peptidase that degrades B chain of insulin, glucagon, and ACTH: purification by affinity chromatography and some properties.

A metallo-endopeptidase that catalyzes at near neutral pH the hydrolysis of certain polypeptides was purified from rat kidney microsomes by a simplified procedure using affinity chromatography on Sepharose 4B coupled with insulin B chain. The purified enzyme showed a single component by chromatography on diethylaminoethyl cellulose and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 213,000. Studies on its substrate specificity showed that the purified enzyme rapidly degrades insulin B chain, glucagon, adrenocorticotropin, and, at a significantly lower rate, insulin A chain. The enzyme has a very weak or no activity toward ribonuclease and vasopressin. In contrast, the enzyme does not degrade denatured hemoglobin, bovine serum albumin, insulin (nano- or micromolar), oxytocin, furylacryloylglycyl-leucine amide (FAGLA), synthetic substrates of cathepsin C (β-napthalamides of glycine-l-arginine and l-histidine-l-serine), or synthetic substrates of aminopeptidases (l-arginine- or l-glutamic acid-β-napthylamide). The enzyme degrades reduced or oxidized B chain at about the same rate, but S-sulfonated B chain is degraded at a markedly lower rate. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. Activity of the enzyme is markedly inhibited by chelating agents (EDTA and o-phenanthroline) and, modestly, by high concentrations of citrate and histidine. Activity of the enzyme is also markedly inhibited by simple thiol compounds (dithiothreitol, glutathione, and mercaptoethanol), but not by sulfhydryl reagents (N-ethylmaleimide or iodoacetate). The inactive apoenzyme, prepared by treatment of the enzyme with EDTA followed by dialysis, was reactivated by Zn²⁺ > Ca²⁺, minimally by Cu²⁺, but not by Hg²⁺. Some anions (phosphate, borate, and bicarbonate) were strongly inhibitory, but chloride had no effect. The following agents were found to have no effect: soybean and lima bean trypsin inhibitors, N^?-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N^α,?-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin (Trasylol), phenylmethylsulfonyl fluoride (a serine protease inhibitor), 1-methyl histidine, 3-methyl histidine, histamine, imidazole, and heparin.     

An f-type thioredoxin fromArabidopsis thaliana Leaves

Thioredoxin, a small redox protein with an active site disulfide/dithiol, is ubiquitous in bacteria, plants, and animals and functions as a reducing agent and modulator of enzyme activity. A thioredoxin has been purified to electrophoretic homogeneity from the leaves ofArabidopsis thaliana using procedures such as DE-52 ion exchange chromatography, Sephadex G-50 gel filtration, Q-Sepharose ion exchange chromatography, and DEAE-Sephadex A-25 chromatography. The purified thioredoxin was determined to be a single band on SDS-PAGE, and its molecular weight was estimated to be 21 KDa, which was much larger than those of most other known thioredoxins. It was proved to be an f-type thioredoxin, since it could activate fructose-l,6-bisphosphatase, but it could not activate NADP⁺-malate dehydrogenase. As a protein disulfide reductase, it could reduce the disulfide bonds contained in insulin. As a substrate, it showed a Km value of 20.2 μM onEscherichia coli thioredoxin reductase, and it had an optimal pH of 8.0. The molecular weight of the purified f-type thioredoxin is not consistent with those of the five divergent h-type thioredoxins already identified by cDNA cloning. The purified f-type thioredoxin is the first example isolated fromA. thaliana.     

Alteration of ribosomal protein S17 by mutation linked to neamine resistance in Escherichia coli: II. Localization of the amino acid replacement in protein S17 from a neaA mutant

A mutant of Escherichia coli strain K12S, nea^R301, resistant to the antibiotic neamine was found to have an altered 30 S ribosomal protein S17. The modification involves a change in the electrophoretic mobility of this protein. S17 proteins wore purified from the mutant and the parental strain, respectively, and the amino acid compositions of all tryptic peptides were compared. The results show that the mutational alteration involves a replacement of histidine by proline in peptide T8 from mutant nea^R301. The amino acid replacement is located at position 30 of the S17 protein chain. We conclude, therefore, that the mutation nea^R301 affects the structural gene for protein S17 (rps Q).     

Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose by Escherichia coli

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB^glc. Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.     

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.     

Evaluation of conformational changes in diabetes‐associated mutation in insulin a chain: A molecular dynamics study

Insulin plays a central role in the regulation of metabolism in humans. Mutations in the insulin gene can impair the folding of its precursor protein, proinsulin, and cause permanent neonatal‐onset diabetes mellitus known as Mutant INS‐gene induced Diabetes of Youth (MIDY) with insulin deficiency. To gain insights into the molecular basis of this diabetes‐associated mutation, we perform molecular dynamics simulations in wild‐type and mutant (Cys^A7 to Tyr or C(A7)Y) insulin A chain in aqueous solutions. The C(A7)Y mutation is one of the identified mutations that impairs the protein folding by substituting the cysteine residue which is required for the disulfide bond formation. A comparative analysis reveals structural differences between the wild‐type and the mutant conformations. The analyzed mutant insulin A chain forms a metastable state with major effects on its N‐terminal region. This suggests that MIDY mutant involves formation of a partially folded intermediate with conformational change in N‐terminal region in A chain that generates flexible N‐terminal domain. This may lead to the abnormal interactions with other proinsulins in the aggregation process. Proteins 2015; 83:662–669. © 2015 Wiley Periodicals, Inc.     

Enzymatic semisynthesis of human insulin: an update

Peptide bond formation can be enzymatically catalysed by the reverse reaction of proteases. Application is seen in the industrial production of human insulin. Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1-29)-A(1-21) insulin precursor as the starting material. This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr-X (X = blocking residue) (two-step reaction) or (2) subjecting Ala-B30 of porcine insulin or Gly-A1 of the single chain insulin precursor to transpeptidation with Thr-X (one-step reaction). Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two-step reaction. Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two-step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture.