B22Asn人胰岛素突变体的研究

通过ＤＮＡ定点突变法将人胰岛素Ｂ２２Ａｒｇ改造成Ｂ２２Ａｓｎ,去除其一级结构中碱性蛋白酶位点,以期提高胰岛素在体内的稳定性,突变体基因克隆到表达载体ＰＢＶ２２０中,在Ｅ．ｃｏｌｉ ＤＨ５α中进行表达,分离纯化后的表达产物经胰蛋白酶和羧肽酶Ｂ联合作用获得重组Ｂ２２Ａｓｎ－人胰岛素,该突变胰岛素具有抗胰蛋白酶水解能力,但是其与受体的结合能力只有标准猪胰岛素的１２．４％,胰岛素结构中的Ｂ２２Ａｒｇ可能在     

B8Gly在胰岛素结构模体中的可能作用

在胰岛素结构模体ｎ１－Ｃｙｓ－Ｇｌｙ－Ｘ１０－Ｃｙｓ－ｎ２－Ｃｙｓ－Ｃｙｓ－Ｘ３－Ｃｙｓ－Ｘ８－Ｃｙｓ－ｎ３中,有７个绝对保守的氨基酸残基,只有位于Ｂ８位的是Ｇｌｙ。通过定点突变将其改变为Ａｌａ,得到「Ｂ８Ａｌａ」人胰岛素,其受体结合能力和体内生物活力分别为天然猪胰岛素的２．５％和１０％。「Ｂ８Ａｌａ」人胰岛素和重组人胰岛素的远紫外圆二色谱比较表明,「Ｂ８Ａｌａ」人胰岛素的α－螺旋的相对含量有一家     

一种重组抗胰蛋白酶单体人胰岛素突变体的设计,制备？…

用缺口双链ＤＮＡ的定向突变方法分别将胰岛素前体中Ｂ链第２２、２８、２９和３０位改变为Ａｓｐ、Ｌｙｓ、Ｐｒｏ和Ｌｙｓ,酵母分泌表达的前体经胰蛋白酶直接酶切,得到重组〔Ｂ２２Ａｓｐ、Ｂ２８Ｌｙｓ、Ｂ２９Ｐｒｏ、Ｂ３０Ｌｙｓ〕人胰岛素。它与受体的结合能力约为猪胰岛素的６％,而体内生物活力保留５０％。通过ＦＰＬＣ分子筛测定其自身结合能力,在生理条件下浓度达１０＾－４ｍｏｌ／Ｌ时它以单体形式存在。作为可抗胰     

[B_1Ala,B_2Ala,B_3Lys]-胰岛素的制备和生物活性(英文)

本文报道了胰岛素分子中Ｂ１～３序列（Ｐｈｅ－Ｖａｌ－Ａｓｎ）为Ａｌａ－Ａｌａ－Ｌｙｓ取代的胰岛素类似物制备及其生物性质。［Ｂ１Ａｌａ,Ｂ２Ａｌａ,Ｂ３Ｌｙｓ］－胰岛素仍保留天然胰岛素的全部体内活性和受体结合能力,但体外促脂肪生成活性和免疫活性分别只为胰岛素的７０％和０．８８％。本文还就胰岛素Ｂ链Ｎ端肽段对其结构和功能的影响进行了讨论。     

胰岛素蛋白质工程;[B9Glu,B10Asp]人胰岛素

用缺口双链ＤＮＡ的定向突变方法分别将胰岛素Ｂ链第９和第１０位的Ｓｅｒ和Ｈｉｓ改变为Ｇｌｕ和Ａｓｐ,获得「Ｂ９Ｇｌｕ,Ｂ１０Ａｓｐ」人胰岛素。其受体结合能力为猪胰岛素的３４．４％,而体内活力与猪胰岛素基本相同     

[B1Ala,B2Al2,B3Lys]—胰岛素的制备和生物活性

本文报道了胰岛素分子中Ｂ１￣３序列（Ｐｈｅ－Ｖａｌ－Ａｓｎ）为Ａｌａ－Ａｌａ－Ｌｙｓ取代的胰岛素类似物制备及其生物性质。［Ｂ１Ａｌａ,Ｂ２Ａｌａ,Ｂ３Ｌｙｓ］－胰岛素仍保留天然胰岛素的全部体内活性和受体结合能力,但体外促脂肪生成活性和免疫活性分别只为胰岛素的７０％和０．８８％。本文还就胰岛素Ｂ链Ｎ端肽段对其结构和功能的影响进行了讨论。     

人胰岛素原类似物(BKRA)基因的合成与表达

为了利用基因工程生产胰岛素,按照已知的人胰岛素Ａ、Ｂ链氨基酸序列和大肠杆菌偏爱的氨基酸密码子设计并合成了人胰岛素原类似物（ＢＫＲＡ）基因,其中以赖（Ｋ）－精（Ｒ）二肽编码区取代人胰岛素原Ｃ肽编码区．为了避免其编码蛋白在大肠杆菌中表达时被降解,通过人工接头将２个ＢＫＲＡ基因串联起来,接头部分氨基酸序列为Ａｒｇ－Ａｒｇ－Ａｓｎ－Ｓｅｒ．将串联的ＢＫＲＡ基因克隆到表达载体ｐＥＴ－２８ａ（＋）,实现了在大肠杆菌中的融合表达,表达产物以包含体形式存在,约占细菌总蛋白２４％．表达产物氨基末端具有六组氨酸肽段,以ＨｉＴｒａｐ凝胶进行亲和层析,一步纯化可达纯度９５％以上．放射免疫测定表明,纯化的融合蛋白具有胰岛素抗原活性．表明已构建成人胰岛素原类似物的高效表达菌株     

天花粉蛋白Y14F/R22L定点突变及其活性研究

利用多聚酶链式反应（ＰＣＲ）技术,对天然天花粉蛋白（ｎＴＣＳ）基因在Ｔｙｒ１４和Ａｒｇ２２两个保守残基处同时进行定点突变,即Ｔｙｒ１４变成Ｐｈｅ,Ａｒｇ２２变成Ｌｅｕ,然后克隆到ｐＥＴ－８ｃ高效表达载体上,构建成重组质粒ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ．经序列分析,定点突变的结果与预先设计的完全一致,突变后的天花粉蛋白命名为Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ．将ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ转化到Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３,ｐＬｙｓＳ）中,进行表达．经ＣＭ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ柱纯化,ＳＤＳ－ＰＡＧＥ鉴定,纯度可达９０％．ＲＩＰ活性测定显示,Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ的活性比ｎＴＣＳ降低了７．５倍,活性变化不显著,因此,ＴＣＳ的Ｔｒｙ１４和Ａｒｇ２２对维持其活性部位构象并不是必需的．但由于Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ在Ｅ．ｃｏｌｉ中的表达量与ｎＴＣＳ相比明显下降,因此,Ｔｙｒ１４和Ａｒｇ２２可能与ＴＣＳ翻译后的折叠有关．     

胰岛素蛋白质工程：高活性（B10Asp）人胰岛素

用基因定位突变方法将胰岛素Ｂ链第１０位的Ｈｉｓ变为Ａｓｐ,获得高活力胰岛素（Ｂ１０Ａｓｐ）人胰岛素,其受体结合能力和离体生物少分别为猪胰岛素的２６２％和２３５％体内生物活力也明显高于猪胰岛素,它的促细胞生长能力为猪胰岛素的１７４％。     

胰岛素蛋白质工程：高活性［B10Asp］人胰岛素

用基因定位突变方法将胰岛素Ｂ链第１０位的Ｈｉｓ变为Ａｓｐ,获得高活力胰岛素──［Ｂ１０Ａｓｐ］人胰岛素。其受体结合能力和离体生物活力分别为猪胰岛素的２６２％和２３５％;体内生物活力也明显高于猪胰岛素;它的促细胞生长能力为猪胰岛素的１７４％。     

一种重组抗胰蛋白酶单体人胰岛素突变体的设计、制备和鉴定

用缺口双链DNA的定向突变方法分别将胰岛素前体中B链第 2 2、2 8、2 9和 3 0位改变为Asp、Lys、Pro和Lys,酵母分泌表达的前体经胰蛋白酶直接酶切 ,得到重组 [B2 2Asp、B2 8Lys、B2 9Pro、B3 0Lys]人胰岛素。它与受体的结合能力约为猪胰岛素的 6% ,而体内生物活力保留 5 0 %。通过FPLC分子筛测定其自身结合能力 ,在生理条件下浓度达 10 -4mol/L时它以单体形式存在。作为可抗胰蛋白酶酶解的单体胰岛素类似物 ,它可能具有一定的应用前景     

B22 Glu Des-B30 Insulin： A Novel Monomeric Insulin

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.     

Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells

Mutations in the human cationic trypsinogen are associated with hereditary pancreatitis. The cDNA coding for human cationic trypsinogen was subcloned into the expression vector pcDNA3. The mutations R122H, N29I, A16V, D22G, and K23R were introduced by site directed mutagenesis. We constructed an expression vector coding for active trypsin by subcloning the cDNA of trypsin lacking the coding region for the trypsin activating peptide behind an appropriate signal peptide. Expression of protein was verified by Western blot and measurement of enzymatic activity. AR4-2J cells were transiently transfected with the different expression vectors and cell viability and intracellular caspase-3 activity were quantified. In contrast to wild-type trypsinogen, expression of active trypsin and mutated trypsinogens reduced cell viability of AR4-2J cells. Expression of trypsin and R122H trypsinogen induced caspase-3 activity. Acinar cells might react to intracellular trypsin activity by triggering apoptosis.     

A fibrinolytic enzyme from a marine green alga, Codium latum

A fibrinolytic enzyme was isolated from a marine green alga, Codium latum, and designated C. latum protease (CLP). It also had fibrinogenolytic activity, hydrolyzing A alpha, B beta and gamma chains with preference in this order. As CLP hydrolyzed oxidized insulin B chain at position Arg22-Gly23, and the peptide map of lysozyme digested with CLP was similar to that with trypsin, CLP would be expected to have a high substrate specificity, similar to that of trypsin. Protease activity peaked at pH 10, and was completely inhibited by diisopropyl fluorophosphate (DFP). Therefore, we conclude that CLP is a trypsin-like serine protease.     

Structure and activity of insulin, XV[1-5]. Further evidence for the importance of arginine residue B22 in the activity of insulin. Semisyntheses of despentapeptide-(B23 - 30)-insulins varied in B22 using desnonapeptide-(B22 - 30)-insulin and tetrapeptides

Insulin hexamethyl ester was digested by trypsin. The resulting desoctapeptide-(B23 - 30)-insulin pentamethyl ester was purified. This compound was digested by carboxypeptidase B to remove the arginine residue B22 at the end of the B chain. Then the N-terminal amino groups of the remaining desnonapeptide-(B22 - 30)-insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the glutamic acid residue B21 of this product was coupled to the following synthetic tetrapeptide esters: Arg-Gly-Phe-Phe-OMe, Lys(Boc)-Gly-Phe-Phe-OMe, Orn(Boc)-Gly-Phe-Phe-OMe, Cit-Gly-Phe-Phe-OMe, Ala-Gly-Phe-Phe-OMe and Gly-Gly-Phe-Phe-OMe. The syntheses of these peptide esters are described. After removal of all protecting groups, despentapeptide-insulin (B22-Arg) and analogues of this product with variation in position B22 could be obtained. They were purified by column chromatography. The biological activities of these components were determined by the mouse fall test. In the case of despentapeptide insulin (C-terminus Arg-Gly-Phe-Phe), the activity rose to the expected value of 34%. The insulin variants with amino acid residues other than arginine in position B22 had much lower activities: with lysine 13%, with ornithine 12%, with citrulline 9%, with alanine 8% and with glycine 6%. Desnonapeptide-insulin by itself posses an activity of 3%. These results demonstrate once more the essential nature of arginine residue B22 for insulin activity.     

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.     

人白介素18(IL-18)RGD模体抗黑色素瘤细胞B16活性研究

利用定点突变及DNA重组技术,在人白细胞介素18(IL-18)的cDNA序列中插入了GGC序列,使IL-18第39精氨酸残基和第40天冬氨酸残基之间插入一个甘氨酸残基,从而构建了RGD模体。此重组的cDNA序列构建入表达质粒pPIC9K,并转化Pichia Pastoris酵母GS115,利用表达系统进行了高效表达。用Sephadex G-100凝胶过滤纯化表达产物,获得初步纯化的蛋白。研究表明,IL-18在体外对黑色素瘤细胞株B16没有作用,而IL-18-RGD抑制作用明显,IC50 = 8.10μmol/L;应用小鼠动物模型研究结果显示,IL-18及IL-18-RGD均对黑色素瘤细胞B16有抑制作用,且IL-18-RGD比IL-18的作用强;同时,对鸡胚绒毛尿囊膜(chicken chorioallantoic membrane,CAM)血管生成的抑制实验发现,IL-18-RGD 对CAM血管生成的抑制作用也比IL-18强。但IL-18-RGD仍保存对PBMC诱导产生IFN-γ能力。结果提示,IL-18-RGD在具有抗炎,抗感染作用的同时增添了抑制肿瘤血管新的功能。     

含RGD模体的人白细胞介素18(IL-18)突变体的酵母表达及其抗血小板聚集活性

利用定点突变及DNA重组技术,在人白细胞介素18(IL18)cDNA序列中插入GGC序列,使IL18第39位精氨酸残基和第40位天冬氨酸残基之间插入一个甘氨酸残基,从而构建了RGD模体.此重组的cDNA序列构建入表达质粒pPIC9K,并转化Pichiapastoris酵母GS115,利用表达系统进行了高效表达.用SephadexG100凝胶过滤纯化表达产物,获得初步纯化的蛋白.对该蛋白进行了血小板聚集抑制实验和对GPⅡbⅢa与Fn结合的抑制实验.含RGD模体的重组IL18(IL18RGD)显示了较强的体外抑制血小板聚集活性,IC50=8.8μmolL;并具有与GPⅡbⅢa的竞争结合活性,IC50=8.0μmolL.该含有RGD模体的重组IL18仍保存对PBMC诱导产生IFNγ能力.结果表明,此IL18RGD嵌合体在具有抗炎,抗感染的同时增添了新的抑制血小板聚集功能.     

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3h post-challenge. After a decrease at 6h, the expression level increased again and reached 207.8-fold at 12h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCfKZSPI-12) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was 173 nmol L(-1). These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response.