A7—B7二硫键缺失对胰岛素原重折叠及结构的影响

为研究A7 B7二硫键在胰岛素原结构和折叠中的作用 ,构建了A7 B7二硫键缺失的胰岛素原突变体 ,研究了其与野生型胰岛素原在体外重折叠产率、自由巯基氧化速度、CD谱、受体及抗体结合活性 ,以及对胰蛋白酶酶切敏感性的差别。结果表明 ,A7 B7二硫键缺失可导致胰岛素原α 螺旋明显减少以及对胰蛋白酶的酶切敏感性显著增加 ,其对胰岛素原结构的影响主要导致了受体结合活性的大幅度降低。突变体在体外重折叠 1h后巯基氧化速率较野生型明显减慢 ,但其最终折叠产率与野生型相当。由此提出一个胰岛素原折叠的可能途径 ,即A链链内二硫键最先形成 ,然后是两对链间二硫键。且A2 0 B19二硫键比A7 B7二硫键很可能先形成 ,在折叠中更重要。     

一种人胰岛素类似物的制备、鉴定与活性研究

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B25和B26位之间插入Glu,构建了［B10 Glu,B25-Glu-B26］胰岛素原融合蛋白基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性、凝胶过滤等步骤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经过DEAE离子交换和RP-HPLC纯化得到胰岛素突变体类似物,并经过质谱测定鉴定.凝胶过滤法测定了蛋白质分子自身的缔合性质,圆二色谱（CD）测定了构象的变化.并分别测定了放免活性、受体结合活性及小白鼠低血糖惊厥实验.结果表明,突变体分子缔合性明显下降.放免活性和受体结合活性分别约为标准胰岛素的63.5%和114.4%, 整体活力略高于天然胰岛素.     

核苷二磷酸激酶A的定点突变及C4S突变体的制备和活性研究

目的:对核苷二磷酸激酶A(NDPK-A)二硫键异构的关键残基C4进行定点突变,构建、表达并纯化C4S突变体,测定其磷酸转移酶活性和DNase活性,研究二硫键异构对NDPK-A活性的影响。方法:以pBV220-nm23-H1质粒作为模板,通过设计合适的引物对NDPK-A进行定点突变,将第4位半胱氨酸突变为丝氨酸,构建NDPK-AC4S突变体;在大肠杆菌BL21中表达,DEAE-sepharose Fas tFlow与Cibacron Blue 3GA Sepharose CL-4B纯化目的蛋白,获得均一重组蛋白,纯度达到98%;DNA序列测定及重组蛋白的肽质量指纹图谱(PMF)分析均证明构建正确突变体;高效液相色谱法(HPLC)与DNA消化法分别测定野生型NDPK-A与C4S突变体的磷酸转移酶活性与DNase活性差异。结果:NDPK-AC4S突变体的磷酸基转移酶与DNase酶活性均高于野生型NDPK-A。结论:NDPK-A缺失二硫键后,活性增高。NDPK-A形成链内二硫键可能是其活性负调控模式之一。     

二硫键稳定的抗CD3/抗Pgp微型双功能抗体的构建、表达及活性测定

向抗CD3/抗Pgp(P-glycoprotein)微型双功能抗体(Diabody)中引入二硫键,使diabody两条链以共价键交联,增强其稳定性。用重叠PCR(Overlap PCR)和PCR方法,将抗CD3/抗Pgp diabody中抗CD3或抗Pgp轻重链可变区的适当部位定点突变成半胱氨酸,进行原核表达。表达产物经阳离子层析柱和抗Etag亲和层析柱分离纯化,还原性和非还原性SDS-PAGE电泳鉴定。应用间接免疫荧光和竞争性免疫荧光结合流式细胞分析技术(FCM)检测生物学活性。将改造前后的两种抗体分别置37oC含0.2%人血清白蛋白(Human serum albumin,HAS)的PBS中孵育,取多个时间点比较两者与两种把抗原的结合能力。基因重组质粒经测序证实序列正确。向抗Pgp轻重链引入突变的diabody(dsPgp-diabody)在原核表达系统中,SDS-PAGE显示纯化后的蛋白二硫键不能配对。而向抗CD3轻重链引入突变的diabody(dsCD3-diabody)能够在原核表达系统中进行可溶性表达,二硫键能够正确配对。并且与改造前的diabody相比,dsCD3-diabody的抗原结合特异性...     

重组单链胰岛素的连接肽与其生物功能的关系

为研究重组单链胰岛素的生物活性与连接肽之间的关系,用基因定点突变的方法分别以二肽A-K,七肽A-A-A-A-A-A-K和十二肽A-A-A-A-A-A-A-A-A-A-A-K连接胰岛素的B30和A1,得到3个单链胰岛素分子PIP,[A]₅PIP和[A]₁₀PIP.它们的受体结合能力分别为胰岛素的0.14%, 14.3% 和11.1%, 体内生物活性与受体结合能力一致,而它们的促生长活性分别为胰岛素的17%,116.3%和38%.结果表明:(ⅰ)单链胰岛素也具有胰岛素的促代谢和促生长功能;(ⅱ)单链胰岛素与胰岛素受体的结合能力与连接肽的长度和氨基酸组成密切相关, 其受体结合能力随连接肽的改变,可由无到100%,进一步说明胰岛素与其受体结合时,B链C端远离A链N端是必需的;(ⅲ)单链胰岛素的促细胞生长能力也与连接肽长度和组成密切相关,且比其自身的促代谢能力强.     

人胰岛素A、B链基因的合成、克隆及其在大肠杆菌中的表达

根据人胰岛素A、B链的多肽顺序,设计并用一种简便快速的多肽基因合成了A、B链基因。合成的A 链和B 链基因分别克隆到pWR590质粒上,构建了表达型质粒pWR590-HIA和pWR 590-HIB,它们能够表达由β-半乳糖苷酶N-端约590个氨基酸残基与A 链或B 链组成的融合蛋白(两者之间由Met 连接)。A 链或B 链融合蛋白经BrCN 降解,磺化及分离纯化等步骤,得到了磺化A、B 链。磺化A、B链体外重组得到人胰岛素。     

抗凝活性肽RGD226基因构建、表达、产物纯化及活性分析

通过PCR法 ,将来源于蛇毒蛋白质eristostatin中的一段含有RGD(Arg Gly Asp)序列的十三肽(SRVARGDWNDDYS)的基因 ,以一个无胰岛素活性但保留天然免疫原性的胰岛素原突变体(PJG4 0 1)基因为模板 ,置换其B2 8和A1位之间的连接肽基因 ,构建成了能展示RGD功能序列的人源化分子 (RGD2 2 6 )的基因 .通过该基因在大肠杆菌中的表达、产物分离纯化 ,得到高纯度RGD2 2 6 .该RGD肽对由ADP诱导的体外人血小板聚集的半抑制浓度IC50 为 2 2 3μmol L .其胰岛素免疫活性是PJG4 0 1的 15 1% ,提示其在一定程度上保留了胰岛素原的免疫原性 .其受体结合活性不到PJG4 0 1的 0 1% ,说明其胰岛素的生物活性基本丧失 .动物实验证实 ,RGD2 2 6在延长小鼠出血时间上具有较明显的作用     

胰岛素B链N端的修饰及其生物活性变化

通过对在大肠杆菌体系中表达及部分加工的粗产物的反相快速蛋白液相色谱的分离,我们得到了Ｌ－Ｍｅｔ＾ＢＯ－人胰岛素原、Ｌ－Ｍｅｔ＾Ｂ－１－Ｌｙｓ＾ＢＯ－人胰岛素原,氨基酸组成分析结果与设想的完全一致,两者的胰岛素受体活性分别为人胰岛素原的６６％及５４％,而胰岛素的放射免疫活性分别降为人胰岛素原的２２％及９％。表明胰岛Ｂ链的Ｎ端对胰岛素分子与其受体的结合有一定的重要性,而更重要的是它非常可能参与组成胰岛     

C-端引入二硫键对黑曲霉木聚糖酶XynZF-2热稳定性的影响

同源比对黑曲霉XZ-3S木聚糖酶基因xyn ZF-2氨基酸序列,模拟构建木聚糖酶三维结构,确定能够提高酶热稳定性的最佳突变位点。在C-端引入二硫键,突变xyn ZF-2 205位点的色氨酸和52位点的丙氨酸为半胱氨酸,获取突变基因T205C-A52C,表达于大肠杆菌BL21(DE3)。酶学性质比较发现,突变酶Xyn ZFT205C-A52C的最适温度为50℃,比原酶Xyn ZF-2提高了10℃;50℃保温5 min,突变酶相对酶活性为55.36%,原酶相对酶活性为32.62%;原酶与突变酶最适p H均为5.0,但相同p H下突变酶的相对酶活性较原酶高;突变酶的p H稳定区间由原酶的5.0~9.0扩大为3.0~9.0。因此,定点突变T205C和A52C在C-端引入二硫键能提高黑曲霉木聚糖酶Xyn ZF-2热稳定性及p H稳定性。     

胰岛素B链N端的修饰及其生物活性变化

通过对在大肠杆菌体系中表达及部分加工的粗产物的反相快速蛋白液相色谱的分离,我们得到了Ｌ－Ｍｅｔ~（ＢＯ）－人胰岛素原、Ｌ－Ｍｅｔ~（Ｂ－１）－Ｌ－Ｌｙｓ~（ＢＯ）－人胰岛素原,氨基酸组成分析结果与设想的完全一致。两者的胰岛素受体活性分别为人胰岛素原的６６％及５４％,而胰岛素的放射免疫活性分别降为人胰岛素原的２２％及９％。表明胰岛素Ｂ链的Ｎ端对胰岛囊分子与其受体的结合有一定的重要性,而更重要的是它非常可能参与组成胰岛素分子的抗原决定簇。     

Effects of deletion and shift of the A20-B19 disulfide bond on the structure, activity, and refolding of proinsulin

To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the alpha-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [Delta A19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.     

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     

Role of disulfide bonds in the structure and activity of human insulin

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness. Deletion of the A6-A11 disulfide bond caused the least perturbation to the structure. In addition, different refolding efficiencies between the three des mutants suggest that the disulfide bonds are formed sequentially in the order A20-B19, A7-B7 and A6-A11 in the folding pathway of proinsulin.     

Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin. Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats

The biological activities of several derivatives of human proinsulin (HPI) containing peptide bond cleavages or peptide deletions in the connecting peptide region were examined in vivo in rats and in several in vitro systems. The two derivatives which were tested in vivo, split (32-33)HPI and des-(64,65)HPI, both demonstrated greater potency in lowering blood glucose than did intact HPI. The receptor binding affinities of split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, des-(33-56)HPI, des-(31,32)HPI, split (32-33)HPI, and split (56-57)HPI were examined in cultured IM-9 lymphocytes, freshly isolated rat adipocytes, and purified rat liver membranes and were compared to the binding of intact HPI and insulin. In these systems, HPI averaged approximately 1% of the activity of insulin. Modification of proinsulin in the connecting peptide region near the A-chain of insulin to form split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, or des-(33-56)HPI resulted in an increase in affinity for receptor binding ranging from 11 to 27-fold over that of intact HPI. In contrast, modifications near the B-chain of insulin to form either des-(31,32)HPI or split (32-33)HPI resulted in roughly a 5-fold increase in affinity, whereas a cleavage within the connecting peptide to form split (56-57)HPI showed only a 2-fold increase in affinity as compared to intact HPI. The biological potencies of these materials were examined in isolated rat adipocytes. At high concentrations (10(-7) M), each derivative produced the same maximal response. At lower concentrations, differences in the relative potencies paralleled the differences in receptor binding affinity previously noted.     

Replacement of disulfides by amide bonds in the relaxin-like factor (RLF/INSL3) reveals a role for the A11-B10 link in transmembrane signaling

The relaxin-like factor (RLF) also named insulin-like 3 (INSL3) consists of two polypeptide chains linked by two interchain and one intrachain disulfide bond. RLF binds to its receptor (LGR8 also named RXFP2) through the B chain and initiates transmembrane communication by activating the adenylate cyclase through the N-terminal region of both chains. Cystine A11-B10 occupies a unique position on the molecular surface just outside the binding region and between the two signaling ports. We have synthesized an RLF analogue in which the disulfide A11-B10 was replaced by a peptide bond and found that cAMP production ceased while receptor binding was not affected. In contrast, replacing the disulfide A24-B22 by a peptide bond reduced potency proportional to the binding affinity and lowered efficacy to 65%, while replacing disulfide A10-A15 by a peptide bond reduced binding affinity to 32% and lowered potency to 7% but maintained 100% efficacy. The exceptional properties of the derivative bearing an A11-B10 isopeptide cross-link suggests that the disulfide has a special role in signal transduction. We propose that disulfide A11-B10 serves as an insulator between the two ports, whereas the amide functionality disturbs the signal transmission complex likely due to changes in polarity. The clear separation between receptor binding and signal activation sites within this small protein permits one to study how the relaxin-like factor initiates the signal on the receptor that induces intracellular cAMP production.     

Effect of disulfide bond on the conformation and anticoagulant activity of an Arg-Gly-Asp motif displayed on a mutant insulin protein framework

Summary Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motifcontaining sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chain and connected with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6–11 disulfide bond within the A chain. The two RGD-containing insulin genes were over-expressed inE. coli, and purified and designated as RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those of expected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC₅₀ of 3 μM, while the latter was 3.5-fold less active. Thein vivo assay also indicated that the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater anticoagulant activity.     

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.     

A conserved histidine in insulin is required for the foldability of human proinsulin: structure and function of an ALAB5 analog

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His(B10) is well defined: although not required in the mature hormone for receptor binding, in the islet beta cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His(B5) is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala(B5)-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala(B5) analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala(B5) and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His(B5) in native insulin. Substantial receptor binding activity is retained (80 +/- 10% relative to the parent monomer). Although the thermodynamic stability of the Ala(B5) analog is decreased (DeltaDeltaG(u) = 1.7 +/- 0.1 kcal/mol), consistent with loss of His(B5)-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His(B5) facilitate alignment of Cys(A7) and Cys(B7) in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.