Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The turkey erythrocyte membrane insulin receptor was solubilized and reconstituted into vesicles composed of either soy or dimyristoyl phosphatidylcholine. Reconstitution with soy phosphatidylcholine provided a lipid environment containing 43% unsaturated fatty acids, as compared with 82% saturated fatty acids in the dimyristoyl phosphatidylcholine preparation. After reconstitution, both species of vesicles were isolated from a 2 to 30% continuous sucrose gradient at a density of 1.071 g/ml. Scatchard analysis of binding data obtained at 15 degrees C revealed that the reconstituted receptor had a greater affinity for [125I]iodoinsulin in the saturated lipid environment (Ke = 0.167 nM-1; K1 = 2.18 nm-1) than in the unsaturated lipid environment (Ke = 0.0162 nM-1; K1 = 0.479 nm-1). Low affinity binding also was increased in the saturated vesicles. These increases were paralleled by a reduction in the number of available insulin binding sites in the saturated lipid environment. There was no difference, however, in the relative affinity of the reconstituted receptor preparations for insulin or proinsulin. Electron microscopy and gel filtration indicated that the binding differences are not due to differences in vesicle size. They also are not due to differences in the orientation of the receptor within the lipid bilayer, for its sensitivity to trypsin digestion was similar in both types of vesicles. Solubilization studies with 1% beta-octylglucoside indicated, however, that the dimyristoyl phosphatidylcholine vesicles incorporated a slightly lesser amount of insulin receptor. Similar results were also observed at 37 degrees C. These results suggest that the membrane lipid environment, especially the degree of unsaturation of the phospholipid fatty acyl chains, can influence the binding properties of the insulin receptor.
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PMID:Lipid effects on the binding properties of a reconstituted insulin receptor. 705 82

The construction of a gene encoding Lys-human proinsulin, its direct expression in E. coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20-30%. After simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1 L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. After separation with DEAE-Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L of fermentation medium.
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PMID:Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor. 748 87

A novel one-step method for enzyme immobilization in composite gels based on the thermally reversible polymer poly (N-vinyl-caprolactam) was developed. Conditions for entrapment of trypsin and carboxypeptidase B in the gels were optimized. After immobilization, 80 and 90% of the original carboxypeptidase B and trypsin activities, respectively, were found to retain. Both immobilizes enzymes were active in a wider pH and temperature range. The granules with entrapped enzymes were successfully applied to obtain human insulin from recombinant proinsulin.
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PMID:[A new method of immobilizing proteolytic enzymes in polymeric hydrogels]. 816 51

Initial studies on the specificity of the multicatalytic proteinase complex (MPC; EC 3.4.99.46) led to the identification of three distinct proteolytic components designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine proteinase inhibitor. The three components cleave the peptidyl-arylamide bonds in the model synthetic substrates, Z-(D)-Ala-Leu-Arg-2-naphthylamide, Z-Gly-Gly-Leu-p-nitroanilide, and Z-Leu-Leu-Glu-2-naphthylamide, respectively. We report here evidence for the presence in the MPC of two additional distinct components, neither of them capable of cleaving the three model substrates. One of these components cleaves the Leu-Gly and the Leu-Ala bonds in the substrates Cbz-Gly-Pro-Ala-Leu-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Leu-Ala-p-aminobenzoate, respectively, and is activated by treatment of the MPC with DCI, N-ethylmaleimide, Mg2+, Ca2+, and low concentrations of sodium dodecyl sulfate and fatty acids. This component is apparently identical with the previously identified DCI-resistant component of the MPC that cleaves preferentially bonds on the carboxyl side of branched chain amino acids in natural peptides including neurotensin and proinsulin [Cardozo, C., Vinitsky, A., Hidalgo, M. C., Michaud, C., & Orlowski, M. (1992) Biochemistry 31, 7373-7380]. It is probably also identical with the component proposed to be the main factor responsible for the caseinolytic activity [Pereira, M. E., Nguyen, T., Wagner, B. J., Margolis, J. W., Yu, B., & Wilk, S. (1992a) J. Biol. Chem. 267, 7949-7955]. The designation "branched chain amino acid preferring" (BrAAP) is proposed for this component. The second component cleaves peptide bonds between the small neutral amino acids Ala-Gly and Gly-Gly in the substrates Cbz-Gly-Pro-Ala-Ala-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Gly-Gly-p-aminobenzoate, respectively. This component is sensitive to inactivation by DCI, N-ethylmaleimide, and organic mercurials, but unlike the BrAAP it is significantly activated neither by Mg2+ or Ca2+ nor by fatty acids or sodium dodecyl sulfate. The designation "small neutral amino acid preferring" (SNAAP) is proposed for this component. Both components are sensitive to inhibition by the peptidyl-aldehydes N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO; calpain inhibitor I) and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO; calpain inhibitor II) but are resistant to inhibition by Z-LLF-CHO, a potent inhibitor of the chymotrypsin-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids. 843 36

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.
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PMID:Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence. 854 27

C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity. 125I labeled Tyr-C-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human pro-insulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed in Escherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.
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PMID:Preparation of Tyr-C-peptide from genetically altered human insulin precursor. 857 43

The kinetics for trypsin cleavage of different fusion proteins, consisting of human proinsulin and two IgG-binding domains (ZZ), were investigated. To achieve simultaneous removal of the fusion tag and processing of proinsulin to insulin and free C peptide, three versions of the ZZ-proinsulin fusion protein were generated, having different trypsin-sensitive cleavage sites, Arg, Lys-Arg or Lys. The ZZ-proinsulin fusion proteins which accumulated as inclusion bodies in Escherichia coli cells were solubilized, refolded and purified by IgG affinity chromatography. The yield of ZZ-proinsulin monomers exceeded 90%. The kinetics for the trypsin cleavage revealed unexpected differences when comparing the three linkers and it was found that the single arginine linker was most efficiently processed. Characterization of the cleavage products by reverse-phase chromatography, mass spectrometry and N-terminal sequencing verified that human insulin and C peptide were generated. The results demonstrate that high yields of native insulin, C peptide and affinity tag can be achieved by simultaneous cleavage of a fusion protein at three different trypsin-sensitive sites in a single step. The implications for production and recovery of various recombinant proteins are discussed.
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PMID:Single-step trypsin cleavage of a fusion protein to obtain human insulin and its C peptide. 861 42

An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).
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PMID:A removable spacer peptide in an alpha-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae. 862 Oct 69

Met-[A6-Ser, A11-Ser]-human proinsulin (Mut-HPI) was prepared in the same way as described previously for Met-human proinsulin (Met-HPI). After trypsin and carboxypeptidase B cleavage and DEAE-Sephadex A25 separation, Met-[A6-Ser, A11-Ser]-human insulin (Mut-HI) and Met-human insulin (Met-HI) were obtained. Their amino-acid compositions are in a good agreement with those expected. Mut-HI keeps full immuno activity, but loses almost all of its receptor binding activity with Met-HI as the control. Some physico-chemical behaviors of Mut-HI have changed to a certain extent. CD studies of mutant insulin demonstrates that the conformation of Met-HI is very similar to that of HI, while that of Mut-HI has altered slightly. The most important observation is that the binding ability of Mut-HI to the receptor has decreased abruptly. These results suggest that though the intra-A chain disulfide bond is deleted, the other two inter-chain disulfide bonds are still correctly paired, and that the intra-A chain disulfide bond is essential for insulin displaying its biological activity.
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PMID:Characteristic, activity and conformational studies of [A6-Ser, A11-Ser]-insulin. 876 30

The potential for the development of an integrated process for production of human insulin and its C-peptide in Escherichia coli has been investigated. Human proinsulin was produced intracellularly in E. coli fused to two synthetic IgG-binding domains (ZZ) derived from staphylococcal protein A. High expression levels (3 g/l culture) of the gene product, which accumulated as inclusion bodies, was obtained. Solubilization of inclusion bodies by oxidative sulfitolysis and subsequent renaturation was performed directly after cell lysis and pellet wash. IgG affinity chromatography was used for efficient recovery of pure proinsulin fusion protein in a single step. Monomers of the proinsulin fusion protein constituted approximately 70%. A single step conversion of the fusion protein into insulin and C-peptide by trypsin and carboxypeptidase B treatment was achieved by engineering the junction between proinsulin and its affinity handle, ZZ. Characterization of the cleavage products by reversed phase chromatography (RPC) verified that human insulin and C-peptide were generated and that the ZZ affinity handle was resistant to cleavage. Human insulin and C-peptide were recovered with high yields by preparative reversed-phase high performance liquid chromatography (RP-HPLC). The potential use of the presented scheme for large-scale production of recombinant insulin and/or its C-peptide is discussed.
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PMID:Integrated production of human insulin and its C-peptide. 886 1


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