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You-Min Feng

Publications and source records attributed to You-Min Feng.

36 records · Page 2Linked to original sources
PubMed2001

Unfolding of Recombinant Single-chain Insulin in Denaturants Containing Thiol Reagents.

Recombinant single-chain insulin (PIP) contains three disulfide bonds. In the presence of denaturants and thiol reagents, the native structure of PIP was disturbed and its native disulfides were shuffled to form a mixture of scrambled isomers which have different degrees of unfolding. In this paper the unfolding degrees of PIP in urea or guanidine hydrochloride containing 0.2 mmol/L 2-mercaptoethanol was analyzed by reverse-phase HPLC and far-UV circular dichroism(CD). The peptide mapping of PIP scrambles demonstrated that PIP had shuffled its native disulfides under the condition we used. Among others, a major non-natural PIP disulfide isomer was purified and its refolding in vitro was investigated. These results show that PIP has only one thermodynamically stable disulfide linkage, and the non-natural disulfide isomers could refold in vitro efficiently to from native PIP. On basis of these, the differences between PIP, IGF-I and insulin on unfolding and refolding were discussed.

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PubMed2000

Relationship between insulin A chain regions and insulin biological activities.

AIM:To study the relationship between insulin A chain regions and insulin biological activities, we designed a series of insulin analogues with changes at A21, A12-18 of C terminal helical region and A8-10 located in the region of A6-A11 intra-chain disulphide bond.METHODS:Insulin A-chain analogues were prepared by stepwise Fmoc solid phase manual synthesis and then combined with natural B-chain of porcine insulin to yield corresponding insulin analogues. Their biological activities were tested by receptor binding, mouse convulsion and immunological assay.RESULTS: A21Ala Ins retains 70.3% receptor binding capacity and 60% in vivo biological activity.DesA13-14, A21Ala Ins and DesA12-13-14-15, A21Ala Ins still have definite biological activity,7.9% and 4.0% receptor binding,and 6.2% and 3.3% in vivo biological activity respectively. A15Asn, A17Pro, A21Ala Ins maintains 10.4% receptor binding and 10% in vivo biological activity. A8His, A9Arg, A10Pro, A21Ala Ins, A8His, A9Lys, A10Pro, A21Ala Ins and A8His, A9Lys, A10Arg, A21Ala Ins have 51.9%, 44.3% and 32.1% receptor binding respectively,50%, 40% and 30% in vivo biological activity respectively, and 28.8%, 29.6% and 15.4% immunological activity respectively.CONCLUSION:A21Asn can be replaced by simple amino acid residues.The A chains with gradually damaged structural integrity in A12-18 helical region and the demolition of the A12-18 helical region by the substitution of Pro and Asn for A17Glu and A15Gln respectively can combine with the B chain and the combination products show definite biological activity, the helical structure of A12-18 is essential for biological activities of insulin. A8-10 is not much concerned with biological activities, but is much more important antigenically in binding to its antibodies, these results may help us design a new type of insulin analogue molecule.

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PubMed2000

Cloning and Expression of Insulin Receptor Ligand-binding Domains.

Insulin receptor is a transmembrane protein consisting of four subunits, that form a heterotetramer(alpha(2)beta(2))with molecular weight of 350 kD. Because the extracellular subunit(alpha)consists of 731 residues and a cysteine-rich domain, it is difficult to express and crystallize such a large ligand-binding subunit, thus hampering further study on "insulin-receptor" complex. Based on the fact that the domains L1 and L2 of the alpha subunit, consisted of 119 and 118 residues, contained the high and low affinity insulin binding sites, respectively, the cDNAs of L1 and L2 were obtained from a human placental cDNA library by PCR. The cDNAs of L1, L2 and L1-(Ala)(10)-L2(designed ten-alanine-connected L1 and L2)were cloned, respectively, into an expression plasmid pET-3a, and E.coli BL21(DE3)transformants with such plasmids were successfully induced to express the goal proteins. The expression products were isolated and purified by the washing and solubilization of inclusion body, gel filtration chromatography and ion exchange chromatography. Each final product displayed a single band, corresponding the purity above 99%, in SDS-PAGE. These products have also been confirmed respectively as the L1, L2 and L1-(Ala)(10)-L2 by DNA sequencing, amino acid composition analysis and N-terminal amino acid sequencing.

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PubMed2000

A Recombinant Monomeric Human Insulin Mutant with Resistance to Trypsin Design, Preparation and Characterization.

By using a gapped duplex DNA method, the four amino acids on the B chain of insulin, i.e. B22Arg, B28Pro, B29Lys and B30Thr, were mutagenized simultaneously into B22Asp, B28Lys, B29Pro and B30Lys, respectively. The recombinant B22Asp B28Lys B29Pro B30Lys human insulin was obtained simply by the treatment with trypsin of the precursor that was expressed in yeast. This human insulin analog had only 6% of receptor binding activity as that of the porcine insulin, but retained 50% of in vivo biological activity, compared with the latter. The self-association ability of the mutant measured through FPLC gel filtration chromatography showed that it was in monomeric state. As a monomeric insulin analog with resistance to the trypsin digestion, this compound may be promising in practical application.

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PubMed2000

Possible Role of B8Gly in Insulin Structural Motif.

In the insulin structural motif n1-Cys-Gly-X10-Cys-n2-Cys-Cys-X3-Cys-X8-Cys-n3, there are seven absolutely conserved amino acid residues, and the only Gly is at position B8. When B8Gly was substituted with Ala by means of site-directed mutagenesis, a mutant insulin, [B8Ala]human insulin was obtained. The receptor binding capacity and in vivo biological activity of the [B8Ala]human insulin were about 2.5% and 10% of native porcine insulin, respectively. The far-UV circular dichroism (CD) spectra of [B8Ala]human insulin and human insulin showed that the relative content of alpha-helix in the mutant somewhat decreased. The results indicate that the B8Gly in the insulin structural motif is unreplaceble.

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PubMed1999

Secretory Expression of Human Insulin in Methylotrophic Yeast Pichia pastoris.

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.

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PubMed1999

The Role of C-Terminus of Insulin B Chain and the Amino Group of B29Lys Side Chain in the Growth Promoting Activities of Insulin.

By growing the mouse mammary tumor-derived cell line GR2H6 in 96-well plates, we have developed an in vitro bioassay for the growth promoting activities of insulin. This bioassay system offers several advantages over currently used alternatives, such as higher sensitivity, better reproducibility and the processing of many samples simultaneously. Using this method, the mitogenic activities of insulin and its analogues were studied. The analogue with elongated C-tenminus of insulin B chain ( B31Lys Ins-NH(2)) had a higher mitogenic activity than insulin (130% of insulin). The mitogenic activities of analogues with B29Lys amino group blocked was one third of those with B29Lys amino group free, indicating that the C-terminal part of B chain and the amino group of B29Lys were important for the growth promoting activities of insulin.

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PubMed1998

Comparison of the Growth Promoting Effects of Serum Transferrins from Different Animals on Mouse Mammary Tumor Cell Line GR2H6.

The growth promoting effects of seven animal serum transferrins from mammalian, aves, reptilia, amphibian and osteichthyes on mouse mammary tumor cell line GR2H6 in serum-free medium were compared by MTT assays. The results indicated that the mitogenic activities of the transferrins from different species were different, and this discrepancy was approximately parallel to their binding capacities with transferrin receptors on human placenta and GR2H6 cells.

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PubMed1997

Characterization and Identification of Recombinant [B18Ile] Human Insulin.

Recombinant [B18Ile] human insulin was obtained from a mutant [B18Ile] PIP purified by transpeptidation. [B18Ile] human insulin can be crystallized and has 82% of receptor binding activity as that of porcine insulin and retains almost the same level of in vivo biological activity comparing with porcine insulin. It is proposed that the B18Val residue may not be involved in the expression of insulin activity.

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PubMed1997

The Transferrins from Amphioxus and the Molecular Evolution of Transferrin.

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.

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PubMed1997

Secretory Expression of Porcine Insulin Precursor in Kluyveromyces lactis and Its Conversion into Human Insulin.

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.

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PubMed1997

An Assay System for the Growth Promoting Activity of Insulin by (3)H-thymidine Incorporation.

In a chemically defined serum-free medium, insulin can stimulate DNA synthesis of the mouse mammary tumor-derived cell line GR2H6 up to 3 - 5 fold above the control group as measured by (3)H-thymidine incorporation. By growing GR2H6 cells in the 24-well and 96-well plates, a bioassay system for the growth promoting activity of insulin has been established. This method has the advantages such as easy operation, less time-consuming and suitable for measuring a large number of samples simultaneously. It may provide a novel model system for studies on the relationships between the growth promoting activity and the structure of insulin.

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PubMed1996

Protein Engineering of Insulin: [B9Glu, B10Asp] Human Insulin.

B9Ser and B10His of the insulin B chain are substituted respectively by Glu and Asp using a gapped duplex DNA approach for site-directed mutagenesis. A mutant insulin-[B9Glu, B10Asp] human insulin was obtained. The receptor binding capacity of the mutant insulin is 34.4% as that of porcine insulin. However, the in vivo biological activity of [B9Glu, B10Asp] human insulin is almost as the same as that of porcine insulin.

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PubMed1996

Two Chain "Insulin/Insulin-like Growth Factor-I" Hybrids.

The two chain "Insulin/Insulin-like Growth Factor-I" hybrids, Ins/IGF-I(8) and Ins/IGF-I(11), were obtained by means of enzymatic semisynthesis, using desoctapeptide insulin (DOI) and the octapeptide and undecapeptide chemically synthesized according to the sequence 22-29 and 22-32 of IGF-I respectively as the starting materials. Comparative studies of the molecules with insulin indicate that the hybrid molecules retain in vivo the full activity of insulin. So the replacement of B27Thr by Asn and B30Ala by Thr, and the exchange of the sequence orders of B25 and B26, B28 and B29, as well as the extension of tripeptide (Gly-Tyr-Gly) at the B30 do not affect insulin activity.

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PubMed1996

Growth Promoting Activity of Chimeric Molecules of "Insulin-Insulin-like Growth Factor-I"

The chimeric molecules of "Insulin-Insulin-like Growth Factor-I", Ins/IGFI(8) and Ins/IGF-I(11) were obtained by means of enzymatic semisynthesis. The growth promoting activity of these chimeric molecules were evaluated with a mouse mammary tumor derived cell line, GR2H6, and compared with that of IGF-I, insulin and desoctapeptide insulin (DOI). Both compounds have lower activities than IGF-I and effects similar to insulin, but are more potent mitogens than DOI. These results indicate that the C-terminus of insulin B-chain is importantly involved in its growth promoting activities.

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PubMed1996

Characterization of the Insulin and Transferrin Receptors of Two Cancer Cell Lines.

The insulin and transferrin receptors of the PA-1 and GR2H6 cell lines were characterized respectively. The optimum pH for the binding of PA-1 cell is 7.0 with insulin and with transferrin is 7.5 8.0. Scatchard plot and Hill plot analysis show respectively the receptor number of 2.97x10(6)/cell and 6.21x10(7)/cell for insulin receptors of PA-1 and GR2H6 with kD of 4.16x10(-7) M and 7.57x10(-7) M, Hill coefficient of 0.78 and 0.82, showing a negative cooperativity; transferring receptors of PA-1 and GR2H6 were found to have respectively receptor number of 1.25x10(5)/cell and 1.40x10(5)/cell, with kD of 1.38x10(-9) M and 1.66x10(-8) M. The Hill coefficient was 1.04 and 0.98, almost no different from 1, so transferrin receptors showed no cooperativity.

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PubMed1996

Protein Engineering of Insulin: [B9 Glutamic Acid] Human Insulin.

B9Ser of insulin B chain was substituted by Glu using site-directed mutagenesis to obtain a fast-acting insulin-[B9Glu] human insulin. The receptor binding capacity and in vivo biological activity of [B9Glu] human insulin are 21% and 40% as those of porcine insulin respectively.

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