Rapid preparation of human insulin and insulin analogues in high yield by enzyme-assisted semi-synthesis

Rose, Keith; Pury, H De; Offord, Robin E.

doi:10.1042/bj2110671

Cited by 24 publications

(18 citation statements)

References 6 publications

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“…The insulin precursor was converted enzymatically into insulin ester via transpeptidation [18–20] in the presence of trypsin and an excess of O-t-butyl-L-threonine t-butyl ester (H-Thr(tBu)-OtBu) acetate salt. Trypsin mediates cleavage of the N-terminal spacer peptide and the Ala-Ala-Lys connecting linker peptide and the transesterification reaction where threonine ester is added to B-29 residue as previously described [6].…”

Section: Resultsmentioning

confidence: 99%

A Simplified and Efficient Process for Insulin Production in Pichia pastoris

Polez

Origi²,

Zahariev

et al. 2016

PLoS ONE

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A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse.

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Section: Resultsmentioning

confidence: 99%

A Simplified and Efficient Process for Insulin Production in Pichia pastoris

Polez

Origi²,

Zahariev

et al. 2016

PLoS ONE

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“…Arg-38 is specifically removed from (1-38) by carboxypeptidase B (Harris, 1978); (39-104) is trun-,ated by a single Edman degradation cycle by the method of Wallace & Corthesy (1986) for (66-104), but with the use of diethyl ether precipitation, instead of ethyl acetate extraction, to free the peptide of small molecule contaminants. Elongation of (1-38) is performed by reverse proteolysis with trypsin: -the equilibrium is shifted by high concentrations of organic solvent and the nucleophile, an amino acid ester (Rose et al, 1983). Stepwise elongation at the N-terminus of (38-104) or (39-104) requires amino acid active esters; for residues other than arginine, we employed N-hydroxysuccinimide esters (Wallace & Corthesy, 1986).…”

Section: Discussionmentioning

confidence: 99%

Semisynthesis of cytochrome c analogues. The effect of modifying the conserved residues 38 and 39

Proudfoot

Wallace

1987

Biochemical Journal

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We have used chemical and enzymic protein engineering techniques to create analogues of the semisynthetic two-fragment complex (1-37).(38-104) of mitochondrial cytochrome c. This complex, unlike the natural product of specific tryptic cleavage, (1-38).(39-104), from which it is prepared, quite closely resembles the parent protein in functional characteristics and is thus a suitable substrate for modifications designed to study structure-function relations. We have replaced the invariant Arg-38 and the conserved Lys-39 with a range of alternative amino acids and have studied the effects on the principal functional parameters. The hydrogen-bonding capacity of Arg-38 is crucial to the stabilization of the bottom omega-loop, while the positive charge of Lys-39 helps maintain the high redox potential by electrostatic effects at the haem iron.

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“…Octadeutero-PheBl-octadeutero-ValB2 human insulin (DHI) and octadeutero-PheBl-octadeutero-ValB2 porcine insulin (DPI) were prepared as described by Stocklin et al (37). Des-AlaB30 porcine insulin (DAI) was obtained by enzymatic cleavage with Achromobacter lyticus protease of DAI-Thr(Bu l )-O-Bu', prepared essentially as previously described (42). The preparation of recombinant human insulin uniformly labeled with 99.4% 15 N (NHI) will be described elsewhere (R.S., J.-F. Arrighi, K. Hoang-Van, F.C., R.E.O., K.R., unpublished observations).…”

Section: Methodsmentioning

confidence: 99%

A Stable Isotope Dilution Assay for the In Vivo Determination of Insulin Levels in Humans by Mass Spectrometry

Stöcklin

Vadas

et al. 1997

Diabetes

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Insulin levels in humans were measured by a new assay, the isotope dilution assay (IDA), based on stable isotope dilution mass spectrometry. A known amount of a deuterated analog of insulin was used as an internal standard and added to the serum samples before sample processing. After isolation by immunoaffinity chromatography and solid phase extraction, followed by a purification step on reversed-phase microbore high-performance liquid chromatography (HPLC), the insulin-containing fraction was analyzed by mass spectrometry. The relative intensity of the signals due to insulin and its deuterated analog in the mass spectrum was used to determine the concentration of insulin in the sample. Using serum samples of 0.5-2.0 ml, we were able to measure insulin levels in the range of 3-1700 pmol/l in several clinical samples from type II diabetic patients. The basal level of endogenous insulin was also determined in two normal subjects and found to be approximately 20 pmol/l. Insulin secretion was followed after the ingestion of 75 g glucose in one healthy volunteer. Finally, the determination of the insulin level of one hemolyzed post-mortem blood sample, for which immunoassays gave inconsistent results, was performed to help forensic investigations. Our results showed a good correlation with standard immunoassay data, except in six samples where much lower values were obtained by our stable isotope dilution assay, suggesting an overestimation of insulin levels by immunoassay in some cases. As it is not subject to immunological interferences by insulin-related compounds, this new assay has a major clinical advantage in that it avoids confusions related to hyperinsulinemia.

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Rapid preparation of human insulin and insulin analogues in high yield by enzyme-assisted semi-synthesis

Cited by 24 publications

References 6 publications

A Simplified and Efficient Process for Insulin Production in Pichia pastoris

A Simplified and Efficient Process for Insulin Production in Pichia pastoris

Semisynthesis of cytochrome c analogues. The effect of modifying the conserved residues 38 and 39

A Stable Isotope Dilution Assay for the In Vivo Determination of Insulin Levels in Humans by Mass Spectrometry

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