Enzymatic semisynthesis of human insulin: An update

Morihara, Kazuyuki

doi:10.1002/jmr.300030502

Cited by 14 publications

(8 citation statements)

References 19 publications

(11 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In other examples (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), successful ligations were performed mostly on preassociated fragments. The advantage of the present approach is that it does not require the ligation fragments to have high affinity for each other prior to coupling.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Subtiligase: a tool for semisynthesis of proteins.

Chang¹,

Jackson²,

Burnier³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

169

155

View full text Add to dashboard Cite

A variant of subtilisin BPN', which we call subtiligase, has been used to ligate esterified peptides sitespecifically onto the N termini of proteins or peptides in aqueous solution and in high yield. We have produced biotinylated or heavy-atom derivatives of methionyl-extended human growth hormone (Met-hGH) by ligating it onto synthetic peptides containing biotin or mercury. Polyethylene glycol (PEG)-modified atrial natriuretic peptide (ANP) was produced by ligating ANP onto peptides containing sites for PEG modifi'cation. We have established the N-terminal sequence requirements for efficient ligation onto proteins, using either synthetic substrates or pools of ifiamentous phage containing Met-hGH with random N-terminal sequences (substrate phage). To facilitate ligations involving proteins with highly structured or buried N termini, a more stable subtiligase was designed that effectively ligates peptides onto Met-hGH even in 4 M guanidine hydrochloride. The use of subtiligase should expand the possibilities for protein semisynthesis and rational protein design.Coupling of synthetic peptides onto a protein or protein fragment is a potentially powerful means of introducing natural or nonnatural constituents to probe and design protein function (for reviews, see refs. 1-3). For example, enzymatic and chemical peptide ligation methods have been used to produce a variety of semisynthetic proteins (4-14). However, in virtually all cases, coupling relied on noncovalent or covalent preassociation or some other special secondary structural feature ofthe fragments to be ligated. Thus, a more general method for the semisynthesis of any protein would be useful.Recently, a variant of the serine protease subtilisin BPN', in which the catalytic Ser-221 was converted to cysteine and Pro-225 was converted to alanine, was shown to ligate dipeptides onto tetrapeptide esters with high kcat values (20 s'1) and with little hydrolysis of the ester substrate or proteolysis of the amide product (15). Here, we evaluate the sequence requirements for efficient peptide ligation using this double mutant of subtilisin BPN', termed "subtiligase," and design more stable variants of it. These studies suggest that subtiligase may be generally useful for site-specific ligation of peptides containing affinity handles, isotopic labels, heavy atoms, or other nonnatural constituents onto the N terminus of proteins or protein fragments.MATERIALS AND METHODS Materials. Enzymes for DNA manipulations were from New England Biolabs or BRL. Streptavidin-horseradish peroxidase (SAHRP) was from GIBCO/BRL, column resins were from Pharmacia, and atrial natriuretic peptide (ANP) was from Bachem. Oligonucleotides were synthesized by the Oligonucleotide Synthesis Group at Genentech.Peptide Synthesis. All peptides were synthesized by standard methods (16). Peptides esterified with glycol-conjugated phenylalanylamide (glc-F-amide) were synthesized as described (15). For ligations onto immobilized supports, peptides were synthesized on 96-well (---0.17 nmol p...

show abstract

Section: Discussionmentioning

confidence: 99%

“…1-3). For example, enzymatic and chemical peptide ligation methods have been used to produce a variety of semisynthetic proteins (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, in virtually all cases, coupling relied on noncovalent or covalent preassociation or some other special secondary structural feature ofthe fragments to be ligated.…”

mentioning

confidence: 99%

Subtiligase: a tool for semisynthesis of proteins.

Chang¹,

Jackson²,

Burnier³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

169

155

View full text Add to dashboard Cite

show abstract

“…Animal insulins extracted from the pancreas tissue were used for therapy from 1922 until 1974, when semisynthetic human insulin became available in limited quantities by modification of animal insulins. [1][2][3][4] This development was based on identification of the structure of human insulin from human autopsy material. Reference material was obtained by total chemical synthesis and by small quantities of purified human insulin extracted from autopsy material.…”

Section: Development Of Human Insulinsmentioning

confidence: 99%

Equivalent Recombinant Human Insulin Preparations and their Place in Therapy

Sandow

Landgraf

Becker

et al. 2015

European Endocrinology

View full text Add to dashboard Cite

Recombinant human insulin was one of the first products of biotechnology. It was developed in response to the need for a consistent and sufficient worldwide supply. Recombinant human insulin replaced the animal insulins and semisynthetic insulins obtained by modification of animal insulins. Bioequivalence studies were required for regulatory approval. Three reference products were independently established during these procedures: Humulin® (Eli Lilly and Co), Novolin® (NovoNordisk) and Insuman® (Sanofi). Numerous brand names have been used during the commercial development of recombinant human insulin formulations. In this review, three current brand names are used for consistent identification. Human insulin for Humulin and Insuman are produced by fermentation in bacteria () and for Novolin in yeast (). The bioequivalence of recombinant human insulin products was investigated in euglycaemic clamp studies. An overview of such bioequivalence studies is provided here. This paper will consider the relevance of human insulin formulations today and their place in therapy.

show abstract

“…The coupling reaction is preferably performed at minimal water conditions, which reverse the enzymatic activity of trypsin from hydrolysis toward synthesis to generate human insulin ester. Because this coupling reaction could hardly happen under the optimal conditions of cleavage reaction [13], the reaction conditions of one-step transpeptidation was 10,20,24, and 48 H of reaction time. RT, reaction time; IP-TR, insulin precursor fusion protein transpeptidation ratio; P1, insulin precursor fusion protein peak; P2, single-chain insulin precursor peak; P3, Thr B30 (tBu)-OtBu human insulin peak; P4, desoctapeptide-B (23-30) -insulin peak; and P5, desoctapeptide human insulin ester peak.…”

Section: Two-step Transpeptidation Of Insulin Precursor Fusion Proteinmentioning

confidence: 99%

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

Liu

Zhou

Tian³

et al. 2014

Biotech and App Biochem

View full text Add to dashboard Cite

Insulin precursor fusion protein expressed in Pichia pastoris is a single-chain protein with a spacer peptide (EEAEAEAEPK) localized at its N-terminal. Currently, the one-step transpeptidation reaction with low yield and high cost is generally employed to convert the insulin precursor fusion protein into human insulin ester. In this study, a two-step transpeptidation reaction was proposed separating the cleavage step from the coupling step so that each reaction was performed under its optimal conditions. Using this method, the total efficiency doubled and the reaction time was shortened compared with the one-step method. In addition, the amount of O-t-butyl-l-threonine t-butyl ester and trypsin dosages were reduced by 50% and 75%, respectively. This two-step transpeptidation strategy was simple and efficient and could be used for the pharmaceutical production of human insulin.

show abstract

Enzymatic semisynthesis of human insulin: An update

Cited by 14 publications

References 19 publications

Subtiligase: a tool for semisynthesis of proteins.

Subtiligase: a tool for semisynthesis of proteins.

Equivalent Recombinant Human Insulin Preparations and their Place in Therapy

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

Contact Info

Product

Resources

About