[B10,22-Asp,B25-Tyr-NH2]-去β链C端五肽胰岛素的合成和性质

本文报道了[B10,22-Asp,B25-Tyr-NH2]-去B链羧端五肽胰岛素的制备及其生物活性。结果表明,这一类似物的生物活力比去五肽胰岛素(DPI)的活力高一倍,但却比Gerald所报道的[B10-Asp,B25-Tyr-NH_2]-DPI的活力低很多,说明后者的高活性可能依赖于分子中B22-Arg的存在。     

Comparison of reaction rates in trypsin-catalysed transamidation of porcine insulin and its B29-arginine analogue

[B29-Arginine]porcine insulin was prepared from des-(B23-30)-insulin and synthetic octapeptide with the aid of trypsin. Comparison of reaction rates in trypsin-catalysed transamidation of this compound and porcine insulin with threonine ether ester showed that this reaction is determined only by conformational effects and structural features of amino acids leaving from and entering into B30, not by the structure and the pKa value of the basic amino acid in B29.     

Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation

Single chain des-(B30) insulin (SCI) has been synthesized from porcine insulin by trypsin in a medium with a low content of water. Trypsin catalyzes an intramolecular transpeptidation reaction in which the glycineA1 residue substitutes the alanineB30 residue, rendering a LysB29 -GlyA1 peptide link between the A- and B-chains of insulin. The insulin derivative has been purified by column chromatography and appears to be homogeneous in HPLC and disc electrophoresis. The structure was proven to be B(1-29)-A(1-21) insulin by proteolysis with Armilliaria mellea protease followed by a few steps of Edman degradation. The electrophoretic mobility indicates that SCI has a more condensed structure than that of insulin. Perfect rhombohedral crystals were obtained under conditions resembling those under which insulin crystallizes in the same form. SCI was devoid of effect in the blood sugar lowering assay in mice, the estimated potency being less than 0.1% of that of insulin.     

Semisynthetic des-(B27-B30)-insulins with modified B26-tyrosine

Semisynthetic des-(B27-B30)-insulins containing modified B26-tyrosine residues were prepared to refine the understanding of the importance of position B26 with regard to biological and structural properties of the hormone. The following shortened insulin analogues were synthesized by trypsin-catalysed peptide-bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides as amino components: des-(B27-B30)-insulin, des-(B27-B30)-insulin-B26-methyl ester, -B26-carboxamide with varying C-terminal hydrophobicity of the B-chain, and [Tyr(NH2)B26]-, [Tyr(NO2)B26]-, [Tyr(I2)B26]-, [D-TyrB26]des-(B27-B30)-insulin-B26-carboxamide containing non-proteinogenic amino acids in position B26. Starting from insulin and an excess of synthetic Gly-Phe-Phe-Tyr-OMe as nucleophile, des-(B27-B30)-insulin-B26-methyl ester--the formal transpeptidation product at ArgB22--was formed in one step. Biological in vitro properties (binding to cultured human IM-9 lymphocytes, relative lipogenic potency in isolated rat adipocytes) of all semisynthetic analogues are reported, ranging from slightly decreased to two-fold receptor affinity and nearly three-fold biopotency relative to insulin. If the C-terminal tetrapeptide B27-B30 is removed, full relative insulin activity is still preserved, while the shortening results in the loss of ability to associate in solution. Only after carboxamidation or methyl esterification of TyrB26 the self-association typical of native insulin can be observed, and the CD-spectral effects in the near UV spectrum related to association and hexamerization of the native hormone are qualitatively reestablished. The results of this investigation underline the importance of position B26 to the modulation of hormonal properties and solution structure of the shortened insulins.     

Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.

Incubation of pig desoctapeptide-(B23-30)-insulin with trypsin in solvent systems consisting of dimethyl sulphoxide, butane-1,4-diol and Tris buffer resulted in the formation of an extra peptide bond between Arg-B22 and Gly-A1 in the DOPI molecule. This DOPI derivative can also be regarded as pig des-(23-63)-proinsulin. The structure of the new, previously unreported, proinsulin analogue was determined on the basis of amino acid analysis, dansylation and digestion with Staphylococcus aureus V8 proteinase. Receptor-binding ability of des-(23-63)-proinsulin was 20% of that of pig desoctapeptide-(B23-30)-insulin and 0.02% of that of pig insulin.     

重组人胰岛素制备过程中转肽工艺的研究

目的：酵母表达体系制备重组人胰岛素的工艺中,转肽反应属于酶促半合成反应,过程复杂,成本高昂,通过实验研究提高反应效率,降低反应成本。方法：通过优化转肽反应中胰蛋白酶和双保护苏氨酸（Thr（But）OBut）的比例,寻找有利的反应条件,为进一步的条件确定奠定基础;从提高转肽反应物的反应效率出发,调整工艺顺序,将一步转肽反应调整为两步转肽反应;以胰蛋白酶和双保护苏氨酸两种反应物,进行综合成本分析比较;同时采用药典方法测定两种不同工艺制备的重组人胰岛素,进行生物活性的比较分析。结果：通过实验研究,一步转肽反应的收率最高可以达到74%,而两步转肽收率的收率最高可以达到90%;两步转肽反应相比一步转肽反应,酶量减少70%,双保护苏氨酸的使用量降低62.5%,同时提高了转肽反应中胰岛素原的反应浓度,达到12.5 mM,有着进一步开发的潜质;生物活性测定两种工艺生产的重组人胰岛素均符合中国药典的要求。结论：两步转肽反应对比一步转肽反应,提高了反应效率,产物的纯度较高,降低了反应的成本,有利于提高国产胰岛素的市场竞争力。     

Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B

Insulin is a polypeptide hormone which is produced by the β‐cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des‐threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des‐threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2–3 at 25 °C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des‐threonine insulin and hence increased the production yield of active insulin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Impaired negative cooperativity of the semisynthetic analogues human [LeuB24]- and [LeuB25]-insulins

Several semisynthetic analogues of human insulin were prepared by enzyme-assisted coupling of synthetic octapeptides to the C-terminal of porcine desoctapeptide insulin. We report the receptor-binding and biological properties of [Leu^B24]- and [Leu^B25]-insulins, one of which has the same sequence as a “mutant” insulin recently found in a diabetic patient (Tager, H. et al.(1979) Nature $\text{28}$:121–125). [Leu^B24]- and [Leu^B25]-insulins had, respectively, 8–12% and 0.9–1.1% of the binding affinity of human insulin, and 11% and 2.7% of its potency in stimulating lipogenesis in isolated rat fat cells. Neither one was an antagonist of the biological effects of native insulin. While the ability of [Leu^B24]-insulin to induce negative cooperativity was clearly impaired, that of [Leu^B25]-insulin was almost abolished. [Leu^B25]-insulin was also a potent antagonist of the negative cooperativity induced by native insulin.     

Structure-function relationships of shortened [LeuB25]insulins, semisynthetic analogues of a mutant human insulin

Replacement of B25-phenylalanine by leucine in the insulin sequence causes marked inactivation. The effect of this sequence variation was studied here in des-(B26-30)-insulin. [LeuB25]des-(B26-30)-insulin and its B25-amide were prepared by trypsin-mediated semisynthesis from N-terminally protected des-(B23-30)-insulin and synthetic tripeptides. The relative lipogenic potency in isolated rat adipocytes was 8.0% for the truncated analogue with a free B25-carboxyl function, and 18.1% for the amidated analogue. Binding to cultured human IM-9 lymphocytes was 4% and 9%, respectively. Thus, both shortened insulins are markedly more active than [LeuB25]insulin. The PheB25----LeuB25 substitution in both the shortened and the full sequence has a moderate effect on the CD spectrum, indicating that the gross main chain conformation is largely retained in both molecules. Independent of the substitution an absolute increase of the circular dichroism is observed upon amidation of the B25-carboxyl group.     

The solution structure of a monomeric insulin. A two-dimensional 1H-NMR study of des-(B26-B30)-insulin in combination with distance geometry and restrained molecular dynamics.

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.     

Self-association of des-(B26-B30)-insulin. The effect of Ca2+ and some other divalent cations

It has been confirmed by sedimentation equilibrium and sedimentation velocity experiments that des-(B26-B30)-insulin does not self-associate at neutral pH. Sedimentation equilibrium experiments at pH 7, 25 degrees C were conducted to investigate the effects of the structurally and physiologically important divalent cations Zn2+, Cd2+, Pb2+ and Ca2+ on the aggregation state of des-(B26-B30)-insulin (pig) in solution. It was found that all of these ions bring about association of this insulin analogue; Zn2+ and Cd2+ to a more marked degree than Pb2+ and Ca2+. The predominant species in solutions containing Zn2+ appear to be hexamers and hexameric aggregates, in those containing Cd2+, species up to and including tetramers, and in those containing Pb2+ and Ca2+, monomers and dimers of des-(B26-B30)-insulin appear to be the only species present. The possible significance of these findings, especially in relation to a role for Ca2+ in the action of insulin, is discussed.     

Activation of Clostridium botulinum type B and E derivative toxins with lysine-specific proteases

Abstract Clostridium botulinum type B and E derivative toxins were activated with lysyl endopeptidase or endoproteinase Lys-C, which splits only the bond involving the carboxyl group of a lysine residue. Type B toxin was more efficiently activated with lysyl endopeptidase; type E toxin was more efficiently activated with trypsin. Type B toxin was split by the lysine-specific protease into 2 fragments of molecular sizes indistinguishable from those induced with trypsin. Type E toxin was split by the same protease into 3 fragments, 2 of which had M _r identical to those obtained with trypsin, the other having an M _r less than that of the heavy chain but greater than that of the light chain. These results attest that both activation and nicking of type B and E derivative toxins are ascribable to cleavage, not of an arginyl, but of a lysyl bond.     

Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin

We have established a procedure for converting porcine insulin into human insulin using a serine protease from $\text{Achromobacter}\text{lyticus}$ M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with $\text{Achromobacter}$ protease. The coupling between DAI and Thr-OBu^t was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBu^t) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBu^t-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.     

去B链羧端七肽人胰岛素的分离纯化及性质研究

在大肠杆菌温度诱导体系中以非融合方式进行去Ｂ链羧端七肽人胰岛素原基因的表达,获得去Ｂ链羧端七肽人胰岛素原,表达产物占细胞总蛋白量的１３％,表达产物经ＳｅｐｈａｄｅｘＧ－５０柱层析分离及胰蛋白酶和羧肽酶Ｂ的酶促转化等步骤,可得到纯度达９４％以上的去Ｂ链羧端七肽人胰岛素,其氨基酸组成与预期值相符,受体活性是标准猪胰岛素的１％．     

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.     

Designing structural-motifs for the preparation of acylated proinsulin and their regiospecific conversion into insulin modified at Lys29 (K29)

Eight proinsulin encoding genes were prepared and their translation products, when treated with a cocktail of trypsin and carboxypeptidase B, analyzed for the following features. One, their ability to undergo facile removal of the N-terminal linker, generating the phenylalanine residue destined to be the N-terminal of the B-chain of insulin, at a rate similar to that involved in the removal of the C-peptide. Two, processing of diarginyl insulin, produced in the latter process, by carboxypeptidase B then needed to be rapid to remove the two arginine residues, Three, both these operations were to be efficient whether the N-terminal methionine was acylated or not. Four, the proinsulin constructs needed to contain a minimum number of sites for acylation. The aforementioned features were monitored by mass spectrometry and the proinsulin derivative containing MRR at the N-terminal and K⁶⁴ mutated to Q⁶⁴, designated as MRR-(Q⁶⁴) human proinsulin [MRR-(Q⁶⁴) hpi] optimally fulfilled these requirements. The derivative was smoothly acylated with reagents of two chain lengths (acetyl and dodecanoyl) to give acetyl/dodecanoyl MRR-(Q⁶⁴) hpi. Acetyl MRR-(Q⁶⁴) hpi, using the cocktail of the two enzymes, was smoothly converted into, acetyl insulin. However, when dodecanoyl MRR-(Q⁶⁴) hpi was processed with the above cocktail, carboxypeptidase B (whether from animal pancreas or recombinant) showed an unexpected specificity of acting on the K²⁹-T³⁰ bond of the insulin derivatives when K²⁹ contained a large hydrophobic acyl group, generating dodecanoyl des-30 insulin.     

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。     

Semisynthetic analogues of insulin. The use of N-substituted derivatives of methionine as acid-stable protecting groups

1. We describe the use of benzyloxycarbonylmethionine and ethoxycarbonylmethionine for the selective protection of the amino groups of glycine-A1 and lysine-B29 of pig insulin. We have used the Edman method to remove residues from the N-terminal and of the B-chain of the N^A1N^B29-di-protected derivatives. The benzyloxycarbonyl group shows slight but noticeable lability in the acid-cleavage step, but the ethoxycarbonyl group remained intact even after five cycles of degradation. 2. We have prepared the following truncated forms of insulin via the di(ethoxycarbonylmethionyl) derivative: des-Phe^B1-insulin;des-(Phe^B1-Val^B2)-insulin; des-(Phe^B1-Val^B2-Asn^B3)-insulin;des- (Phe^B1-Val^B2-Asn^B3-Gln^B4)-insulin; des-(Phe^B1-Val^B2-Asn^B3 -Gln^B4-His^B5)-insulin. 3. Insulin was re-synthesized from the di-protected des-Phe^B1-insulin by reaction with an active ester of t-butoxycarbonyl-l-phenylalanine. The product after deprotection crystallized, and the immunoreactivity of the crystalline material was identical with that of the native protein. 4. We have prepared the following analogues of insulin in a similar manner: [l-Ala^B1]insulin; [l-Val^B1]insulin; [l-Tyr^B1]insulin; [m-F-l-Phe^B1]insulin; [o-F-l-Phe^B1]-insulin; [o-F-l-Phe^B2]des-Phe^B1-insulin. All had between 34 and 62% of the activity of insulin in the fat-cell test. 5. We have also investigated the use of the benzyol, toluene-p-sulphonyl, p-nitrobenzyloxycarbonyl and 2,4-dinitrophenyl groups for the N-protection of the methionine active esters. Each should have had some particular advantage over the benzyloxycarbonyl and ethoxycarbonyl groups, but all proved in practice to have disadvantages that more than outweighed anything in their favour.     

Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.

Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.     

Use of Bacillus brevis for synthesis and secretion of Des-B30 single-chain human insulin precursor

A synthetic gene encoding a single chain human insulin precursor [B-chain (1-29)-A-chain] linked to the C-terminal lysine of human epidermal growth factor (1-28) (EGF-SCI) was constructed. This gene was expressed using Bacillus brevis. EGF-SCI was isolated from the supernatant of the culture broth. Treatment of EGF-SCI with lysyl endopeptidase resulted in the formation of des-B30 human insulin. The identification of the formed des-B30 human insulin was made by the measurement of molecular weight and amino acid analysis. The binding coefficient to anti-human insulin antibody was comparable to that of human insulin.