用改进的寡核苷酸诱导突变法改造人胰岛素前体基因

我们用改进了的寡聚核苷酸诱导突变法,将两个单一限制性内切酶位点引入人胰岛素前体B链与C链连接处附近,及C链与A铁连接处附近。用含U模板法将B链第30个残基密码子ACC改成ACG,从而引入MluⅠ位点。用修改了的缺口双链DNA突变法,将A链第4个残基密码于GAG改成CTG,引入了一个PstⅠ位点。突变效率的为17％-36％。这个突变体在人胰岛素前体结构及蛋白质折叠的研究中,将有利于更换不同的C-肽。     

Zinc and insulin metabolism

A review of experimental studies of the effect of zinc nutrition on insulin metabolism is presented. In addition to a short introduction to the synthesis, secretion, and action of insulin, the effects of zinc deficiency—specifically on glucose tolerance, insulin secretion, insulin synthesis and storage, and on total insulin-like activity—are dealt with. The concentrations of zinc and chromium in serum, pancreas, and liver are compared to those of zinc-deficient animals and pair-fed controls. In contrast to pair-fed controls, zinc-deficient rats had unaltered proinsulin contents after glucose stimulation, but they showed a diminished glucose tolerance, lowered serum insulin content, and an elevated total insulin-like activity. The serum zinc concentration of the deficient animals was greatly reduced and did not change during glucose stimulation, whereas it rose in the case of the pair-fed controls. The serum chromium concentration increased in both groups in response to glucose stimulation. In the pancreas of the deficient animals, the zinc concentration was reduced 60% and it increased during the glucose tolerance test. In the liver there were no significant differences. The chromium concentrations were elevated in both the pancreas and liver of the zinc-deficient rats by 60 and 100%, respectively, and were not influenced by glucose injection. These studies show clearly that nutritional zinc deficiency influences insulin metabolism and action.     

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

Inhibition of Platelet Aggregation of a Mutant Proinsulin Chimera Engineered by Introduction of a Native Lys-Gly-Asp-containing Sequence

An eight amino acid sequence, CAKGDWNC, from disintegrin barbourin, was introduced into an inactive human proinsulin molecule between the B28 and A2 sites to construct a chimeric, anti-thrombosis recombinant protein. The constructed Lys-Gly-Asp (KGD)-proinsulin gene was expressed in Escherichia coli and then purified. The KGD-proinsulin chimera protein inhibits human platelet aggregation, induced by ADP, with an IC₅₀ value (molar concentration causing 50% inhibition of platelet aggregation) of 830 nM and demonstrates also specific affinity to glycoprotein IIb/IIIa receptor. Its insulin receptor binding activity remaines as low as 0.04% with native insulin as a control.     

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.     

Increased production of low molecular weight recombinant proteins in Escherichia coli.

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-Xaa-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.     

In vitro assessment of cord blood–derived proinsulin-specific regulatory T cells for cellular therapy in type 1 diabetes

Background

Antigen-specific regulatory T cells (Tregs) have proven to be effective in reversing established autoimmunity in type 1 diabetes (T1D). Cord blood (CB) can serve as an efficient and safe source for Tregs for antigen-specific immunomodulation in T1D, a strategy that is yet to be explored. Therefore, we assessed the potential of CB in generation of proinsulin (PI)-specific Tregs by using HLA class II tetramers.

Met-Lys-双C肽人胰岛素原基因的构建表达及分离纯化

应用 Ｐ Ｃ Ｒ 定点突变方法构建编码 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原基因,并在大肠杆菌中以包含体方式获得表达 表达产物经还原、重组、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 分离纯化,获得 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原,经胰蛋白酶与羧肽酶 Ｂ的酶解, Ｒｅｓｏｕｒｃｅ Ｔ Ｍ Ｑ 阴离子交换柱层析分离制备得人胰岛素,其放免活性、受体结合活性均与猪胰岛素相同      

In vitro folding of methionine‐arginine human lyspro‐proinsulin S‐sulfonate—Disulfide formation pathways and factors controlling yield

We investigated the in vitro folding of an oxidized proinsulin (methionine‐arginine human lyspro‐proinsulin S‐sulfonate), using cysteine as a reducing agent at 5°C and high pH (10.5–11). Folding intermediates were detected and characterized by means of matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS), reversed‐phase chromatography (RPC), size‐exclusion chromatography, and gel electrophoresis. The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI‐MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI‐MS analysis of Glu‐C digested, purified intermediates indicated that an intra‐A‐chain disulfide bond formed first among A6, A7, and A11. Various non‐native intra‐A (A20 with A6, A7, or A11), intra‐B (between B7 and B19), and inter‐A‐B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non‐native intra‐A and intra‐B bonds. At a cysteine‐to‐proinsulin‐SH ratio of 3.5, all intermediates with the non‐native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling, which was the slowest step. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine‐to‐proinsulin‐SH ratio, some intermediates and folded MR‐KPB‐hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Reducing protein concentration, while keeping an optimal cysteine‐to‐protein ratio, can improve folding yield significantly. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Specific binding of the C-peptide of proinsulin to cultured B-cells from a transplantable rat islet cell tumor

Specific binding of the C-peptide of proinsulin was evaluated using a transplantable NEDH rat islet cell tumour predominantly composed of insulin-secreting B-cells. Cultured tumour B-cells exhibited greater than 90% viability assessed by trypan blue exclusion, and retained the ability to form tumours with accompanying hypoglycaemia and hyperinsulinaemia after reimplantation. During binding experiments with synthetic rat C-peptide I and iodinated tyrosylated rat C-peptide I, turnout B-cells exhibited 54±6% specific binding. Displacement of tracer increased with increasing concentrations of unlabelled rat C-peptide I (0.25–1,000 ng/ml), and the specificity of binding was substantiated by reduced displacement with human C-peptide. Scatchard analysis of specific C-peptide binding revealed a curvilinear plot with upward concavity. The demonstration of specific C-peptide binding to insulin-secreting B-cells provides evidence for a physiological role of proinsulin C-peptide.