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In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

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.

An aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester was synthesized with aspartic acid replaced by asparagine and peptide bond replaced by ester bond. The aspartic acid of aspartame could be replaced by asparagine as reported in the literature. In this analogue, the hydrogen of amide group could still form a hydrogen bond with the oxygen of ester bond and the ester bond was isosteric with peptide bond. However, the product was not sweet, showing that the peptide bond could not be replaced by ester bond. The peptide C-N bond behaves as a double bond that is not free to rotate and the C, O, N and H atoms are in the same plane. The replacement of peptide bond by ester bond destroyed the unique conformation of peptide bond, resulting in the loss of sweet taste.

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.

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form.

Summary: [B12Thr]human insulin and [Bl2Leu]human insulin were obtained by means of site-directed random mutagenesis. [B12Thr]human insulin retains total biological activity but [B 12Leu]human insulin has much lower biological activity. Receptor binding activities of [B 12Thr]human insulin and [B 12Leu]human insulin are 56% and 3%, respectively, as that of native porcine insulin. The results suggest that the hydrophobic property of the residue side chain at B12 may not be necessary. The three-dimensional structure of insulin shows that in the monomer the side chains ofB12Val, B16Tyr, B24Phe and B25Phe form a hydrophobic surface [12]. This hydrophobic surface was proposed to be a key constituent part of the receptor binding site of insulin[3-6]. It is interesting to study the role of B 12Val in the structure and function of insulin since it is a constituent residue of the hydrophobic surface and it is also conserved in insulins from different species[7]. Schwartz and Hu reported that insulin analogs, in which BI2Val was substituted by Asn, Phe or cz-aminoisobutyric acid by means of chemical synthesis, had much lower biological activities than native insulin [8-9]. Similarly when B 12Val was substituted by Ile or Glu, the biological activity of these analogs was greatly reduced [10]. For further understanding the role of the residue at position B 12 in the receptor binding site of insulin, we have prepared, by means of sitedirected random mtagensis, two mutants of porcine insulin precursor(PIP) in

B24 and B25 phenylalanines (Phe) play important roles in insulin structure and function. Insulin analogs with B24 Phe or B25 Phe replaced by biphenylalanine (Bip) were prepared by enzymatic semisynthesis. The biological activities were determined by receptor binding assay and in vivo mouse convulsion assay. The results showed that B25 Bip insulin has 139% receptor binding activity and 50% in vivo biological activity, whereas B24 Bip insulin is inactive, when compared with native insulin, suggesting that B24 Phe is crucial for insulin activity. The structures in solution were studied by circular dichroism and fluoremetry, and our results suggested that the insulin analogs with low activities tend to be more tightly packed. The association properties were studied by size exclusion chromatography. The Bip-amide replacement of B24 Phe in deshexapeptide insulin or B25 Phe in despentapeptide insulin will cause the monomeric B24 Phe-amide deshexapeptide insulin or B25 Phe-amide despentapeptide insulin to associate and form dimers, whereas the mutations of B24 Phe in insulin will make insulin dimers dissociate into insulin monomers.

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.

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

The methylotrophic yeast Pichia pastoris is a recently developed host for heterologous protein expression. As nonconventional yeast, it has the following advantages over S. cerevesiae: readiness of high-density growth and scaling-up, and the presence of a powerful methanol-regulated promoter AOXl (expressing AOX to 30% of total cell protein) [ 1]. Furthermore, the transformed strains with integrative expression are stable over generations. This system has been used in the expression of many heterologous proteins. We have already expressed insulin precursor in S. cerevisiae [2] and K. Iactis [3]. In order to increase the expression level, we have tried to express it in this system.

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.

An aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester was synthesized with aspartic acid replaced by asparagine and peptide bond replaced by ester bond. The aspartic acid of aspartame could be replaced by asparagine as reported in the literature. In this analogue, the hydrogen of amide group could still form a hydrogen bond with the oxygen of ester bond and the ester bond was isosteric with peptide bond. However, the product was not sweet, showing that the peptide bond could not be replaced by ester bond. The peptide C-N bond behaves as a double bond that is not free to rotate and the C, O, N and H atoms are in the same plane. The replacement of peptide bond by ester bond destroyed the unique conformation of peptide bond, resulting in the loss of sweet taste.

In this paper, we report the semisynthesis of B27 Lys destripeptide insulin (B27 Lys DTrI), i.e. destetrapeptide insulin with an additional Lys residue at the C-terminus of B-chain. B27 Lys DTrI is also monomeric as shown by gel filtration. Its in vivo biological activity is 80% in comparison with that of native insulin. The addition of a Lys residue at the C-terminus of B-chain makes it possible to obtain monomeric B27 Lys DTrI from a precursor expressed in Saccharomyces cerevesiae by tryptic hydrolysis instead of the less efficient tryptic transpeptidation.

The porcine insulin precursor (PIP) gene and its derivative form sp-PIP gene, which had a nona-peptide (called spacer peptide, sp) added at the 5' terminus of PIP gene, were inserted into the plasmid pPIC9 of Pichia pastoris to obtain secretory plasmid pPIC9/PIP and pPIC9/sp-PIP, respectively. P.pastoris GS115 was transformed by pPIC9/PIP or pPIC9/sp-PIP and the high-copy strains, P39(-sp) and S51(+sp), were selected by dot-blotting. The expression levels of PIP and sp-PIP were 10 mg/L and 40 mg/L in 1 L shake flask, respectively, indicating that the spacer peptide could increase the expression. The expression level of PIP (sp-PIP) in P.pastoris was higher than that of PIP in S.cerevisiae and K.lactis reported in this laboratory. The expression level of sp-PIP was 250 mg/L in 10 L fermentor. Recombinant human insulin was obtained by means of transpeptidation of PIP or sp-PIP. The receptor binding capacity is identical with that of porcine insulin. In vivo biological activity of the recombinant human insulin is 27 IU/mg.

A3 Val is important for insulin activity. It is invariant in insulins from different species studied thus far. Based on the three dimensional structure of insulin, it was thought to be involved in receptor binding. Its replacement by Leu resulted in remarkable lowering of insulin activity, indicating the crucial requirement of the side chain geometry at this position. When A3 Val was replaced by Thr, which is hydrophilic but isosteric with Val, substantial insulin activity was retained. Therefore, the isosteric requirement for receptor binding at this site is more stringent than the hydrophobic requirement. Here we report the replacement of A3 Val of porcine insulin by the unnatural allo-Thr. The in vivo biological activity of A3 allo-Thr insulin is similar to that of A3 Thr insulin or native insulin, but its receptor binding activity is 7.6% instead of 50% for A3 Thr insulin, indicating that at the A3 position the hydrophilic OH group of Thr could be more tolerated in receptor binding than the OH group of allo-Thr. The retention of insulin activity by substituting A3 Val with the unnatural isosteric allo-Thr demonstrates again the importance of isosteric interaction in the binding of insulin with its receptor.

Single-chain insulin/IGF-1 hybrid-[Ins/IGF-1(C)], single-chain porcine insulin precursor-(PIP), and B10Asp PIP were prepared by protein engineering. Their growth-promoting activities in mouse breast cancer cell line GR2H6 are 10, 0.2, and 2 times that of insulin, respectively, and 29%, 0.6%, and 6% of that of IGF-1, indicating that the C domain and 9Glu of IGF-1 are important for its growth-promoting activity. Given these results and previous reports, we propose that the C domain, 9Glu, and 23Phe-26Asn beta bend are involved in the growth-promoting functional region of IGF-1.

An amphioxus transferring was isolated and purified from the Xiamen amphioxus. Its physio-chemical properties are the same as those of Qingdao amphioxus transferrin. It is a glyco-protein with a molecular weight of 26 kD that is about a quarter of that of the transferrin from vertebrates. Its partial amino acid sequence and its C-terminal fragment were determined. By comparison with the sequence of human serum transferrin, it has been found that the sequence of human serum transferrin can be divided into 4 fragments with about equal size as the sequences of the amphioxus transferrin. There is quite a high homology between the sequences of amphioxus transferrin and each of the 4 fragments as well as between the four fragments. The results demonstrate that the current transferrin may be evolved from an ancestor transferrin with one quarter of the molecular weight as a result of twice gene duplication. Amphioxus transferrin may be the representative from of such ancestor transferrin.

Porcine insulin precursor (PIP) was cloned to vectors derived from plasmid pKD1 and expressed in Kluyveromyces lactis. The secretory expression level of PIP was 20 to 30 mg per liter of the culture medium. Human insulin obtained from PIP through tryptic transpeptidation was characterized. Its amino acid composition, crystalline shape and biological activity are identical with those of native insulin.

The enzymatic synthesis of amide bonds catalyzed by penicillin acylase is investigated both in H2O solution and in organic solvents containing reverse micelles. The specificity of the reaction is rather high on the side of the acyl component, practically only phenylacetic acid gives sizeable yields. On the contrary, a variety of amino-acid esters, dipeptides, and tripeptides can be used as amino component, e.g., serine methyl ester, methionine ethyl ester, tyrosine ethyl ester, Gly–Asp, Ala–Tyr, Gly–Tyr–Gly etc. However, Many other amino-acid residues do not react, and the possible reasons for this are discussed. Yields varyin rang the 10–80%. A. systematic study to optimize yields by varying the solvent composition is presented for one model reaction. The enzyme is also able to couple certain D-amino-acid residues (e.g. D-methionine ethyl ester or Gly-D-Asp) though at lower rate. Reverse micelles formed by the cationic surfactant cetyltrimethylammonium bromide (CTAB) in CHCl3/isooctane are used to host penicillin acylase and to perform amide synthesis in which the product is perferentially soluble in the organic solvent mixture. The reaction is studied as a function of pH and certain micellar parameters, e. g. wo (wo = [H2O]/[CTAB]). A new membrane enzyme reactor is utilized to separate the product from the enzyme-containing micelles. The adavantges and the limits of this approach are discussed.