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      

人胰岛素原C肽的合成及专一性抗体制备

利用固相多肽合成中的Boc途径合成了人胰岛素原C肽 ,经TSK柱层析一步纯化 ,产物达HPLC及毛细管电泳均一 ,蛋白质序列分析和质谱分析符合理论值 ,总回收率高达 41 %。为了提高寡肽抗体的专一性 ,我们将丙烯酰化的C肽经催化聚合形成了以聚丙酰为骨架 ,C肽为侧链的聚合体 ,分子量约为 2 5kD。免疫新西兰大白兔 1月 ,获得专一性抗体。用酶联免疫吸附法测得抗血清滴度达 2 .5× 1 0 4 ,与BSA无交叉反应。聚丙酰C肽抗原是制备C肽抗体的一种新的尝试 ,而且也可能成为新的多肽工程疫苗之一 ,为其他传染性疾病多肽疫苗的研究提供新的途径     

从基因工程小C肽人胰岛素原类似物（B—R2—A）制备人胰岛素

以融合蛋白的形式,在Ｅｃｏｌｉ中经温度诱导表达了小Ｃ肽人胰岛素原来似物（Ｂ－Ｒ２－Ａ）,表达的融合蛋白可占细胞总蛋白６８％。经磺酸解,及初步分离Ｓ－磺酸型融合蛋白,再经ＣＮＢｒ裂解后,进行还原重组,ＨＰＬＣ分离纯化等步骤,每升发酵液可得到Ｂ－Ｒ２－Ａ约５０ｍｇ。经酶促转化及ＤＥＡＥ－Ｓｅｐｈａｄｅｘ－Ａ２５纯化,得重组人胰岛素约２０ｍｇ,基氨基酸组成与人胰岛素相同,并具有与猪胰岛素相同的生物活性。     

从基因工程小C肽人胰岛素原类似物（B－R2－A）制备人胰岛素

以融合蛋白的形式,在Ｅ．ｃｏｌｉ中经温度诱导表达了小Ｃ肽人胰岛素原类似物（Ｂ－Ｒ２－Ａ）．表达的融合蛋白可占细胞总蛋白６８％．经磺酸解,及初步分离Ｓ－磺酸型融合蛋白,再经ＣＮＢｒ裂解后,进行还原重组,ＨＰＬＣ分离纯化等步骤,每升发酵液可得到Ｂ－Ｒ２－Ａ约５０ｍｇ。经酶促转化及ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５纯化,得重组人胰岛素约２０ｍｇ,其氨基酸组成与人胰岛素相同,并具有与猪胰岛素相同的生物活性．     

用胰酶转肽的方法使人胰岛素原转成人胰岛素

猪胰岛素胰酶转肽成人胰岛素的相似条件下,基因工程生产的人胰岛素原可以50％的产率转成人胰岛素,所得人胰岛素的氨基酸组成与理论值相符,并具有天然活力。     

hGH-双精氨酸C肽人胰岛素原基因的克隆表达及纯化研究

旨在提高基因重组人胰岛素在大肠杆菌中表达的稳定性及表达包涵体蛋白的复性水平.在人胰岛素原N端前融合人生长素N端的一段序列来充当前导肽,同时将C肽设计为两个精氨酸,分10段合成长链寡核苷酸链,利用重叠延伸PCR技术(SOE PCR)扩增得到该基因片段.与表达载体PET-30a连接,转化E.coli BL21(DE3),IPTG诱导表达.表达的融合蛋白采用Ni-NTA亲和层析纯化,纯化后的蛋白经复性、冻干等步骤后用胰蛋白酶,羧肽酶B双酶切再过DEAE Sepharose Fast Flow阴离子交换柱,收集洗脱峰.对制备所得的胰岛素用SDS-PAGE,Western blot进行性质鉴定,及皮下注射小鼠测定生物活性.结果显示,目的蛋白在大肠杆菌BL21(DE3)中得到了表达,表达产物以不溶性包涵体形式纯在,约占大肠杆菌总蛋白的30%.经Ni-NTA亲和层析得到的重组蛋白纯度为85%,DEAE Sepharose Fast Flow阴离子交换纯化得到单组分胰岛素.Western Blot显示制备所得的胰岛素具有胰岛素免疫原性,皮下给药注射小鼠活性测定表明具有明显的降血糖活性.获得了一种高效生产基因重组人胰岛素的方法,为研究胰岛素类似物奠定了前期基础同时也为今后探索胰岛素的非注射给药途径提供了原料.     

胰岛素原C肽多聚体基因在大肠杆菌中的表达及重组C肽在水溶液中的稳定性

合成了胰岛素原C肽三聚体的基因,并在大肠杆菌中得到高效表达。表达的融合蛋白质通过设计的特异性位点的酶切得到重组的人胰岛素原C肽单体。融合蛋白质以可溶性蛋白质的形式表达,表达量约为80mg／L。Ni—NTA亲和柱可有效地从细胞裂解的上清液中分离纯化融合蛋白质,得到纯度大于70％的融合蛋白质37．5mg／L。融合蛋白质可经胰蛋白酶/羧肽酶B双酶切有效释放天然的C肽。释放的C肽经氨基酸组成分析、与标准对照的RP—HPLC分析和免疫发光法定量证实与天然人C肽完全相同。C肽经RP-HPLC纯化后可得,总的收率为1．5mg/L,纯度大于95％。考察了C肽在冻干过程中及在水溶液中的稳定性。结果表明C肽在冻干过程中稳定,在水溶液中其稳定性受pH和温度的影响。在pH3和pH7．4缓冲液中,37℃或70℃下,C肽的降解符合一级动力学。C肽冻干品直接溶解于pH9的缓冲液中,立即降解,降解产物在37℃和70℃下基本稳定。C肽冻干品直接溶解于pH3的缓冲液中,立即发生降解反应,随后可观察到随温度升高和时间延长,降解反应的进行,37℃、10h,可观察到约80．3％的C肽保持,而在70℃、10h,只有43％C肽保持。C肽在pH7．4最稳定,37℃、6h或10g／L BSA存在下,70℃、3h,未观察到降解产物。PH7．4、37℃、10h,在有和没有10g／L BSA存在的情况下,C肽的保持率分别为99％和96％,从而显示了BSA的保护作用。     

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。     

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris

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. Key words proinsulin; C-peptide; Pichia pastoris     

人胰岛素原C肽的表达、纯化及其细胞活性研究

近年来研究发现人胰岛素原C肽 (简称C肽 )有多种生物活性 ,在治疗糖尿病的并发症中有潜在的应用价值。在前期研究中 ,完成了多拷贝串联C肽基因的构建和融合表达 ,在原核中获得了高表达。用发酵的方法大规模表达融合蛋白C肽 (FCP) ,由Ni-NTA柱亲和纯化获得的蛋白经胰蛋白酶和羧肽酶B的酶解及RP HPLC分离获得了纯度超过 95 %的C肽单体。细胞活性研究显示它对U2 5 1、2 93细胞的生长具有一定的促进作用     

Amide 1H n.m.r. study of the folding of ribonuclease C-peptide

The folding of ribonuclease A 1–13 (C-peptide) in H₂O near 0°C has been monitored by means of the amide and side chain NH proton resonances. The C-peptide carboxylate at low temperature forms, in a significant amount, a folded structure similar to the one that the 1–19 S-peptide adopts in the same conditions (3–13 α-helix). A quantitative comparison between helix stabilities of the lactone and carboxylate forms of C-peptide and S-peptide is reported. It is concluded that the proposed His 12⁺ … Hse 13 (COO^? salt bridge, which competes with the one-turn stabilizing salt bridge His 12⁺ … Glu 9^? in the C-peptide carboxylate, does not suppress helix formation as previously suggested but it merely reduces its stability. The behaviour of the N⁵-H resonance of the Arg 10⁺ side chain provides evidence for its implication in a further stabilizing interaction, most probably with Glu 2^?.     

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.     

Proinsulin C-peptide as an alternative or combined treatment with insulin for management of testicular dysfunction and fertility impairments in streptozotocin-induced type 1 diabetic male rats

Diabetes mellitus (DM) is closely associated with male infertility and sexual dysfunction. Recent data indicate that the proinsulin C-peptide (CP) exerts important physiological effects and shows the characteristics of an endogenous peptide hormone. So, this study was done to investigate the effect of C-peptide with or without insulin treatment on testicular function and architecture in diabetic rats. Rats were divided into the following groups: control, diabetic, and diabetic groups treated with either CP alone or combined with insulin. Tested parameters included, estimation of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and glucose levels, testicular samples for histopathology and estimation of malondialdehyde (MDA), total antioxidant capacity (TAC), and B-cell leukemia/lymphoma-2 (BCL-2) levels as well as sperm count and motility. Results showed that DM caused a severe alteration in hormonal profile and reduced sperm parameters along with increased MDA and decrease in both TAC and BCL-2 levels. CP alone or with insulin treatment efficiently reversed all the negative effects of DM on rat testes, with maximum improvement in the combined regimen. Proposed mechanisms may involve its hypoglycemic, antioxidant, and antiapoptotic properties. Thus, CP could substitute for or better combined with insulin to prevent or retard diabetic-induced testicular dysfunction.     

人胰岛素原基因在大肠杆菌中高效外分泌表达

将胰岛素原基因融合到金色葡萄球菌蛋白Ａ的基因上,构建成大肠杆菌中基因融合的外分泌表达载体。它能高效表达且有效地分泌表达产物。利用亲和层析能方便地从培养液中分离出融合蛋白。融合蛋白经ＣＮＢｒ裂解后,经反相ＨＰＬＣ分析,分离得到具有天然结构的胰岛素原并进行了鉴定。