Expression of monomeric insulin precursor in Pichia pastoris and its conversion into monomeric B27 Lys destripeptide insulin by tryptic hydrolysis

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition,and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.     

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。     

Monomeric destetrapeptide human insulin from a precursor expressed in Saccharomyces cerevisiae.

Destetrapeptide insulin (DTI, human insulin with B27-30 removed) was obtained from a monomeric precursor (MIP) expressed in Saccharomyces cerevisiae through tryptic transpeptidation in the presence of synthetic tetrapeptide Gly-Phe-Phe-Tyr. The in vivo biological activity of DTI, determined by mouse convulsion assay, is 22 IU/mg. Its binding activity with insulin receptor on human placental membrane is 80% and its in vitro biological activity, determined by free fat cell assay, is 77%. Compared with native insulin, DTI molecules do not associate in solution but exist in the monomeric form, thus leading to its rapid utilization in vivo.     

B27K—去B链C端三肽胰岛素的制备、生物活力与自聚合性质

液相合成五肽Gly Phe Phe Tyr Lys(Boc)Obut,与B链C端去八肽胰岛素酶促缩合得到B2 7K 去B链C端三肽胰岛素(B2 7K DTrI,B2 7K destripeptideinsulin)。用小鼠惊厥法和小鼠降血糖法测得B2 7K DTrI的整体生物活力为标准胰岛素的 80 % ,B2 7K DTrI与人胎盘细胞膜胰岛素受体结合能力为标准胰岛素的 ( 12 5± 13 ) %。用凝胶过滤法证明B2 7K DTrI自聚合性质降低 ,具有与去B链C端五肽胰岛素相同的单体性质。在B2 7K DtrI结构中 ,B2 7T被K取代 ,其优点是在酵母中表达其前体后 ,可以很方便地通过胰蛋白酶水解获得     

Isolation and chemical characterization of a novel insulin-related neuropeptide from the freshwater snail, Lymnaea stagnalis.

A novel molluscan insulin-related peptide (MIP) III, has been isolated from alcohol extracts of the neurohaemal area of the cerebral neuroendocrine light-green neurones of Lymnaea stagnalis. MIP III was purified by sequential high-performance gel-permeation chromatography followed by reverse-phase HPLC. MIP III is a heterodimer connected by disulphide bonds. Edman degradation analysis and subsequent alignment with the A and B chains of the previously identified MIP I and II showed that the 24-amino-acid peptide with the sequence pQSRPSIVC(E)CCFNQCTVQ(E)LLAYC represents the MIP III A chain, and the 37-amino-acid peptide sequence TTQHTCSILSRPHPRGLCGSTLANMVQWLCSTYTTSS the B chain. The overall amino acid sequence of MIP III shows about 50% similarity with those of MIP I and II, and only 20-40% similarity with other peptides of the insulin superfamily. Important structural features, e.g. disulphide bridges and the hydrophobic core, are conserved in MIP III.     

Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.     

Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr and B27Thr, buried in the dimer of insulin, were identified by means of alanine-scanning mutagenesis. The receptor binding activity, in vivo biological potency and self-association properties of the seven single alanine human insulin mutants were determined. Four of the seven single alanine mutants, [B12Ala]human insulin, [B16Ala]human insulin, [B24Ala]human insulin and [B26Ala]human insulin, are monomeric insulin, which indicates that B12Val, B16Tyr, B24Phe and B26Tyr are crucial for the formation of insulin dimer. The monomeric [B16Ala]human insulin and [B26Ala]human insulin retain 27 and 54% receptor binding activity, respectively, and nearly the same in vivo biological potency compared with native insulin, so they could be developed as the fast-acting insulin.     

Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum.

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.     

Preparation and activity of nitrated insulin dimer

Nitration of insulin using tetranitromethane causes polymerisation involving cross-linked tyrosyl residues. By performing this reaction with insulin crystals, in which it is known that B16 tyrosine of one monomer is closely associated with B26 of the neighbouring monomer within the dimer, it has been possible to isolate a covalent dimer of insulin cross-linked between these two tyrosines. It was, however, first necessary to block the reactive A14 tyrosine. Both rhombohedral (hexameric) and cubic (dimeric) pig insulin crystals were used, the latter proving successful in yielding a pure dimeric product as shown by oxidative sulphitolysis and HPLC. The purified nitrated dimer was biologically active (ca. 10% potency compared to monomeric insulin in a lipogenesis assay) suggesting that the residues responsible for insulin's action are present on the surface of the dimer and not buried in the interface.     

一种筛选单体速效胰岛素的简便方法

通过基因突变方法制备的单体速效胰岛素Lispro Insulin已上市用于治疗糖尿病,如何利用简便快速的方法研究获得新的单体速效胰岛素成为研究的热点。以Lispro Insulin为模型,利用猪胰岛素的胰蛋白酶酶切大片段(DOI,去B链C端八肽胰岛素)和化学合成的八肽,通过胰蛋白酶的酶促合成方法为筛选新的单体速效胰岛素提供了新的途径。结果显示,酶促合成得到的95％纯度的Lispro Insulin具备了单体速效胰岛素的不自身聚合的特点。     

Changes in secondary structure follow the dissociation of human insulin hexamers: a circular dichroism study

Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2-zinc human insulin, zinc-free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [B/S9D,T27E] human insulin, show significant differences. The secondary structure analysis of the 2-zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% beta-strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10-15% increase in antiparallel beta-structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2-zinc, zinc-free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two-state processes.     

Preparation of [B23-D-alanine]des-(B25-B30)-hexapeptide-insulin by a combination of enzymic and non-enzymic synthesis.

Des-(B25-B30)-hexapeptide-insulin with B23-glycine replaced by D-alanine was prepared by a combination of enzymic and non-enzymic syntheses. The purified product was homogeneous in polyacrylamide-gel electrophoresis and could be crystallized. The biological activity in vivo of crystalline [B23-D-Ala]des-(B25-B30)-hexapeptide-insulin was determined as 58% of that of standard pig insulin (27 i.u./mg).     

Characterization of a cDNA clone encoding molluscan insulin-related peptide II of Lymnaea stagnalis

A cDNA clone encoding molluscan insulin-related peptide (MIP) II was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP-I cDNA). The MIP-II cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, C alpha and C beta, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP II and I differ remarkably in primary structure; in contrast, the C alpha peptide domains are fully identical. MIP II has only limited sequence similarity with insulins and related peptides. Both MIP II and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and II genes are expressed in a single type of neuron: the growth-controlling neuroendocrine light green cells of the Lymnaea CNS.     

B22 Glu Des-B30 Insulin： A Novel Monomeric Insulin

Studies on monomeric insulin with reduced self-association are important in the development of insulin pharmaceutical preparations with rapid hypoglycemic action on patients with diabetes. Here we report a novel monomeric insulin, B22 Glu des-B30 insulin, prepared from a single chain insulin precursor with B22 Arg mutated to Glu, which was expressed in Pichia pastoris and converted to B22 Glu des-B30 insulin by tryptic digestion. It still retains 50% of the in vivo biological activity of porcine insulin and does not form a dimer even at a concentration of 10 mg/ml, showing that B22 Glu plays a key role in reducing the self- association of the insulin molecule without greatly reducing its biological activity. This novel monomeric insulin might have potential applications in the clinic.     

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing.

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

A monomer-dimer model explains the results of radiation inactivation: binding characteristics of insulin receptor purified from human placenta

The technique of radiation inactivation has been used on highly purified human placental insulin receptor in order to determine the functional molecular size responsible for the insulin binding and to evaluate the "affinity regulator" hypothesis, which has been proposed to explain the increase in specific insulin binding to rat liver membranes observed at low radiation doses [Harmon, J. T., Hedo, J. A., & Kahn, C. R. (1983) J. Biol. Chem. 258, 6875-6881]. Three different types of inactivation curves were observed: (1) biphasic with an enhanced binding activity after exposure to low radiation doses, (2) nonlinear with no change in binding activity after exposure to low radiation doses, and (3) linear with a loss in the binding activity with increasing radiation exposures. A monomer-dimer model was the simplest model that best described the three types of radiation inactivation curves observed. The model predicts that an increase in insulin binding activity would result after exposure to low radiation doses when the initial dimer/monomer ratio is equal to or greater than 1 and a monomer is more active than a dimer. The monomer size of the binding activity was estimated to be 227,000 daltons by this model. This value most likely reflects the size of the monomeric alpha beta form. To substantiate this model, the purified receptor was fractionated by Sepharose CL-6B chromatography. The insulin binding profile of this column indicated two peaks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flexibility and bioactivity of insulin: an NMR investigation of the solution structure and folding of an unusually flexible human insulin mutant with increased biological activity

The structure and folding of a novel human insulin mutant, [Thr(B27) --> Pro, Pro(B28) --> Thr]insulin (PT insulin), in aqueous solution and in mixtures of water and 2,2,2-trifluoroethanol (TFE) have been studied by NMR spectroscopy. It was found that PT insulin has a highly flexible structure in pure water and is present in at least two different conformations, although with an overall tertiary structure similar to that of native insulin. Furthermore, the native helical structures are poorly defined. Surprisingly, the mutant has a biological activity about 50% higher than native insulin. In contrast, in TFE/water solution the mutant reveals a propensity of forming a well-defined structure at the secondary structure level, similar to monomeric native insulin. Thus, as shown by a detailed determination of the structure from 208 distance restraints and 52 torsion angle restraints by distance geometry, simulated annealing, and restrained energy minimization, the native insulin helices (A2-A7, A13-A19, and B10-B19) as well as the beta-turn (B20-B23) are formed in 35% TFE. However, the amount of tertiary structure is decreased significantly in TFE/water solution. The obtained results suggest that only an overall tertiary fold, as observed for PT insulin in pure water, is necessary for expressing the biological activity of insulin, as long as the molecule is flexible and retains the propensity to form the secondary structure required for its receptor binding. In contrast, a compact secondary structure, as found for native insulin in solution, is unnecessary for the biological activity. A model for the receptor binding of insulin is suggested that relates the increased bioactivity to the enhanced flexibility of the mutant.     

Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase

The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.     

Semisynthetic sheep des-A1-glycine insulin (author's transl)]

The synthetic Des-1-glycine-A-chain of sheep insulin as the monomeric cyclic bisdisulfide and native bovine B-chain bissulfonate were reduced together with mercaptoethanol. They combined at pH 10.6 to yield Des-A1-glycine-insulin. This was purified by gel and ion exchange chromatography. The low insulin activity (0.4 - 0.6%) as measured by the fat cell test as well as the change in the CD spectrum indicated that the loss of the N-terminal glycine of the A-chain results in fully inactive insulin. This confirms the results obtained earlier by partial synthesis of Des-A1-glycine-insulin.