人LL-37与干扰素α2a密码子的优化及其在毕赤酵母中的融合表达

通过密码子优化,在毕赤酵母中高效表达人LL-37与IFN-α2a融合蛋白。先按P.pastoris密码子偏好性对LL-37与IFN-α2a的原始密码子进行了改造,并在二者之间加上Gly Gly Gly Gly Ser的柔性连接接头,人工合成设计的新序列,最后通过p PIC9K载体将其整合入P.pastoris GS115的基因组,构建出重组菌株GS115LI。利用遗传霉素浓度梯度筛选出两株高拷贝的GS115LI1和GS115LI2菌株。对这两株菌经发酵诱导后的发酵液进行SDS-PAGE检测、抗病毒活性检测和抗菌活性检测,证明重组株既能够成功表达出LL-37与IFN-α2a的融合蛋白,而且该融合蛋白成功保留了抗菌肽与干扰素的功能活性。诱导发酵后,融合蛋白的产量可达到819.1 mg/L,经盐析、疏水层析和离子交换层析分离,可得到纯度达97%的融合蛋白产物,回收率可达到46.2%,纯化后产品的效价可达2.6×108 IU/mg。经过密码子优化后,成功实现在毕赤酵母中高效表达出既有LL-37抗菌活性又具有干扰素α2a活性的融合蛋白。     

hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究

为进一步改造重组人激肽释放酶1(hK1),以期提高其生物活性,制备了通过柔性接头相连接的重组人激肽释放酶1-L-IgG1 Fc融合蛋白(hK1-L-Fc)。采用重叠延伸PCR技术构建hK1-L-Fc融合基因,克隆至表达载体pcDNA3.1,在中国仓鼠卵巢细胞(CHO-S)中表达。利用Protein A 亲和层析柱纯化融合蛋白,SDS-PAGE、Western blotting、飞行时间质谱(MALDI-TOF-MS)、HPLC检测表达产物,底物法检测融合蛋白的体外活性。结果显示:成功构建pcDNA3.1-hK1-L-Fc重组表达载体;获得稳定表达融合蛋白的细胞株;无血清悬浮批式培养的表达量在0.7 mg/L以上;纯化的蛋白其纯度在95%以上,分子量约60 kDa;活性检测显示其比活性在9.2 U/mg以上,较hK1-Fc蛋白提高了18%以上。     

人甲状旁腺素(1-34)衍生物在大肠杆菌中表达、纯化及其生物学活性

为研究Gly hPTH(1 34)衍生物的生物学活性 ,用重叠PCR方法合成编码hPTH(1 34)的DNA片段 ,克隆到融合表达载体pGEX 2T的缩短型谷胱甘肽转移酶基因GST6 9△的 3′末端 ,构建正确读码框架的融合基因 .在两个基因间引入蛋白质羟胺切割位点序列 ,转入E .coliJM10 9中 ,IPTG诱导表达 .该融合蛋白的表达量占菌体总蛋白的 2 0 %以上 ,主要以包涵体形式存在 ,盐酸羟胺切割表达产物 .分析表明 ,80 %左右的融合蛋白被裂解为GST6 9△和Gly hPTH(1 34) .经分子筛柱层析和反相层析分离纯化获得重组Gly hPTH(1 34)衍生物 ,纯度达 98%以上 ,回收率约为 10mg/升发酵液 ,分子量为 4 177,等电点 (pI)为 8 4 0 ,N端 16个氨基酸 ,除第一个为甘氨酸外 ,其余与天然hPTH(1 34)序列一致 .Western印迹结果表明 ,Gly hPTH(1 34)衍生物具有hPTH(1 34)的免疫学活性 .体外活性测定结果表明 ,Gly hPTH(1 34)衍生物能刺激人成骨细胞HOSTE85增殖、增加细胞内胶原合成、ALP活性增高和cAMP生成量增加 ,并呈量效关系 ,提示它具有与化学合成的hPTH(1 34)相同的生物学活性 ,N端多一个Gly对其活性无明显影响 .     

毕赤酵母高密度发酵表达血管紧张素转化酶C-结构域

血管紧张素转化酶（ACE, EC3.4.15.1）在调节血压方面具有重要作用。研究证实,ACE的C结构域（ACE-C）是使血管紧张素I (AngI)分解的主要活性位点。在5 L 发酵罐中, 对重组毕赤酵母表达ACE C-结构域的发酵工艺进行优化,探讨温度、pH、甲醇浓度等主要因素对重组蛋白表达量和酶活力的影响。结果表明,当工业培养基添加2%蛋白胨为氮源时, ACE C-结构域的降解现象得到了有效控制;采用诱导温度为26℃,pH5.5,甲醇含量为1.5%的表达条件,ACE C-结构域表达量和酶活力分别达到446 mg/L和38.2U/ml,比活力达到86U/mg,是Sigma公司ACE标准品比活力的2倍,为大规模制备ACE C-结构域蛋白,筛选专一性更强的ACE C-结构域抑制剂奠定了基础。     

His-tag对胆固醇氧化酶基因在大肠杆菌中表达的影响

为考察组氨酸标签(His-tag)对Brevibacterium sp.DGCDC-82中胆固醇氧化酶基因(ChoAb)在大肠杆菌中表达的影响,将PCR扩增后得到的结构基因与pET28a(+)连接,构建重组质粒pETChoAb(不带His-tag),pETChoAbn(His-tag位于N端)和pETChoAbc(His-tag位于C端)并在大肠杆菌中进行表达.对重组酶进行酶活检测,结果表明His-tag位于ChoAb的C端和N端,COD单位体积酶活由未带标签时的1.72 U/mL分别提高到4.03 U/mL和11.36 U/mL.利用软件Quantity One对SDS-PAGE电泳条带进行灰度分析,结果显示与不带His-tag的COD相比,His-tag位于ChoAb的C端和N端,COD表达量由8.8％增加到16.4％与72.3％.同时菌体浓度分别提高了1.2倍和3.2倍.作为纯化标签,该研究结果对His-tag用于诊断用酶COD的分离纯化可以提供一定的理论指导.     

重组鲑鱼降钙素前体多肽的制备及其性质研究

硫氧还原蛋白的第 38位 Met突变为 Ala的 Trx- s CT- Gly基因在 E.coli BL2 1 ( DE3)中得到高效表达 .用 Thio Bind亲和树脂纯化表达的融合蛋白 .结果说明 38位的 Met突变为 Ala不影响融合蛋白与树脂的特异性结合 ,融合蛋白的纯度达 90 %以上 .在含有 3mol/L尿素的 70 %甲酸中 ,室温 48h,至少 80 %融合蛋白被溴化氰裂解开 .采用 CM-纤维素吸附 ,用稀盐酸解吸附 ,得到纯度为92 %的降钙素前体多肽 s CT- Gly.氨基酸的序列分析结构表明 ,重组 s CT- Gly的 N端 1 0个氨基酸与预期一致 .在强酸性条件下 ,没有发生氨基酸的脱酰胺反应 ,氨基酸组成分析与预期基本一致 .质谱法测定的分子量为 3492 ,毛细管电泳测定的等电点 p I为 6.46,大鼠降钙素比活性为 1 90 IU/mg左右 ,与天然的人降钙素相当 .     

核酸酶P1的原核表达、纯化及酶学特性分析

为了建立一种核酸酶P1(Nuclease P1,NP1)的原核表达纯化系统,首先采用重叠延伸PCR将22段寡核苷酸拼接,获得人工合成的NP1基因。将其克隆至分泌型表达载体pMAL-p4X获得重组质粒pMAL-p4X-NP1,然后将重组载体转化T7 Express和Origami B(DE3)菌株诱导表达,利用Amylose亲和层析柱纯化获得重组蛋白,并对其活性、热稳定性和金属离子依赖性进行系统分析。SDS-PAGE结果显示,重组蛋白MBP-NP1(Maltose binding protein-NP1)在T7 Express和Origami B(DE3)菌株中均可表达,且以可溶性形式存在。活性检测表明Origami B(DE3)菌株中获得的重组蛋白活性高于T7 Express菌株(75.48 U/mg:51.50 U/mg);利用蛋白酶Factor Xa切除MBP标签后,两种重组蛋白的比活力均有提高,分别为258.13 U/mg和139.20 U/mg。重组NP1表现出良好的热稳定性,80℃温浴30 min后重组酶仍具有90%以上的活力。2.0 mmol/L Zn2+对NP1有比较明显的激活作用,相同浓度的Cu2+则对该酶有强烈的抑制作用。该研究实现了NP1在大肠杆菌系统中的功能性表达,为NP1纯酶的制备提供一个替代途径。     

胰高血糖素样肽-1融合蛋白在原核系统中的表达

[目的]构建点突变的GLP-1Gly8,并与人血清白蛋白(HSA)结构域Ⅰ融合,延长GLP-1的半衰期。[方法]采用常规PCR扩增白蛋白结构域Ⅰ片段,利用SOE-PCR扩GLP-1Gly8基因并将两个基因拼接,得到的HSA-GLP-1Gly8融合基因经Bam HⅠ和XhoⅠ双酶切后连接到p ET30a表达载体,重组质粒p ET30a-HSA-GLP-1Gly8转入E.coli BL21(DE3)宿主菌中进行IPTG诱导表达。[结果]PCR扩增分别获得140 bp的GLP-1Gly8基因499 bp的HSA的片段及经融合后的HSA-GLP-1Gly8基因。表达载体p ET30a-HSA-GLP-1Gly8在E.coli BL21(DE3)宿主菌中经IPTG诱导,过表达了分子量约为22 k Da的融合蛋白。[结论]成功构建了p ET30a-HSA-GLP-1Gly8原核表达载体,融合蛋白在BL21(DE3)菌中以包涵体的形式过表达。     

来源于嗜热真菌Talaromyces leycettanus JCM12802的类膨胀素基因鉴定及功能探究

挖掘新颖的类膨胀素基因,丰富类膨胀素基因资源,探究其功能,加深我们对类膨胀素及其作用机制的认识,促进其工业应用。通过RT-PCR的方法从嗜热真菌Talaromyces leycettanus JCM12802中克隆得到了一个1 196 bp的类膨胀素基因Tlexlx1,并与大多数真菌来源的膨胀素相比,Tl EXLX1缺少一个N端的CBM结构域。Tl EXLX1与来源于Penicilliopsis zonata的假定蛋白ASPZODRAFT_140583具有最高的序列相似性81%,与Penicillium digitatum Pd1来源的Expansin-like protein 1相似性为56%。同时构建野生型Tl EXLX1和含有Tl SWO1的N端CBM区的突变型CBM-Tl EXLX1重组质粒并在毕赤酵母中表达纯化,并对其基本性质进行分析。该基因含有1个内含子(83 bp),编码370个氨基酸和一个终止密码子。Tl EXLX1推导氨基酸序列包括一个22个氨基酸的N端信号肽序列,一个类GH45结构域和一个类膨胀素结构域。结果表明,重组蛋白Tl EXLX1和融合蛋白CBMTl EXLX1具有较高的葡聚糖酶活性(地衣多糖:7.2 U/mg和17.2 U/mg;大麦葡聚糖:4.4 U/mg和9.4 U/mg)和微弱的微晶纤维素水解活性。以地衣多糖为底物时,二者的最适作用温度和pH一致(60℃和6.0)。Tl EXLX1可以破坏微晶纤维素整齐光滑的表面结构,与商业纤维素酶有一定的协同效果。获得新型的类膨胀素蛋白,有一定的水解活性,在木质纤维素降解等工业中存在潜在的应用价值。     

拟南芥LFR原核重组蛋白纯化和多克隆抗体制备

拟南芥中有一类含ARM结构域的蛋白质,研究表明它们中的一些在植物的生长发育和激素应答等方面发挥着重要的作用.在拟南芥突变体筛选中,获得了一个推测编码蛋白含ARM重复序列的新基因突变体lfr(leaf and flower related mumnt),它在叶子和花的发育过程中表现出较明显的表型.为进一步研究该基因编码蛋白的生物学功能及其分子作用机制,构建了pGEX-2TGST:LFR融合蛋白重组表达载体,将重组质粒转化到工程菌中诱导表达菌体蛋白,经SDS.聚内烯酰胺凝胶电泳检测,结果表明,融合重组蛋白成功获得了高效表达,分子质量在77 ku左右.重组蛋白经谷胱甘肽S.转移酶(GST)标签蛋白亲和层析法纯化,SDS-PAGE制备胶割胶富集,电洗脱法纯化后得到纯度较高的抗原.经对新西兰兔进行5次免疫,获得了多克隆抗血清.采用免疫吸附方法对抗血清进行了纯化,结果得到只识别LFR重组蛋白的抗血清.进一步提取拟南芥野生型及突变体的核蛋白,经蛋白质印迹检测,结果显示,在分子质量50ku左右处出现特异的蛋白质条带,证明所制备的抗血清可以与拟南芥LFR蛋白特异性结合.     

大鼠脂多糖结合蛋白的分离纯化及对脂多糖的调节作用

经硫酸铵沉淀、Bio Rex70树脂的阳离子交换层析和MonoQ预装柱的阴离子交换层析 ,从大鼠急性期血清中分离纯化了脂多糖结合蛋白 .SDS PAGE为单一条带 ,分子量 60kD ,所纯化的大鼠脂多糖结合蛋白可明显增强脂多糖与单核巨噬细胞的结合 .脂多糖结合蛋白对脂多糖诱导肺泡巨噬细胞中肿瘤坏死因子α(TNF α)mRNA表达的调节作用具有明显的剂量依赖性 .     

多串心钠素的纯化与活性测定

为获得纯化心钠素(ANP)单体,采用离子交换及疏水柱层析,纯化融合蛋白麦芽糖结合蛋白(MBP)-ANP和MBP-3ANP,用凝血因子Xa切割MBP-ANP后,经阳离子柱分离获得ANP单体.对ANP单体与BMP-3ANP进行生物学活性检测.1　材料与方法1.1　材料含心钠素多拷贝基因的重组表达质粒pMal-nANP...     

猪繁殖与呼吸综合征病毒N蛋白的原核表达、纯化与结构分析

将猪繁殖与呼吸综合征病毒 (PRRSV)BJ 4毒株N基因克隆至原核表达载体pET2 8a中 ,得到重组表达载体pET2 8 N ,转化EscherichiacoliBL2 1(DE3)细胞 ,获得可溶性表达 ,表达量占菌体蛋白的 2 8%。经ProbandNi2 亲和层析获得重组蛋白P2 8 N ,圆二色谱 (CD)测定结果表明 ,P2 8 N重组蛋白螺旋占 2 6 1% ,折叠占 2 3 7% ,转角 19 8% ,卷曲占 30 3%。并进一步绘制出PRRSVN蛋白的二级结构图     

A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest

The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at 99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was 相似文献   

Isolation of a cDNA encoding a protease from Perinereis aibuhitensis Grube

The cDNA encoding a protease of Perinereis aibuhitensis Grube （PPA） was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21（DE3） and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.     

The purification of orotidine-5'-phosphate decarboxylase from yeast by affinity chromatography.

We have prepared an affinity column for the purification of orotidine-5'-phosphate decarboxylase from yeast. The column effects a 3200-fold purification from yeast homogenate in one pass; simple additional steps produce enzyme that has been purified 6700-fold and is not contaminated by any other protein that can be detected by sodium dodecyl sulfate-acrylamide gel electrophoresis. Overall, 35% of the activity present in the yeast is recovered as pure enzyme. The resin for the column is synthesized by attaching the ethylenediamine amide of 5-(2-carboxyethyl)-6-azauridine 5'-phosphate to carboxymethyl-agarose.     

Purification of streptomycin adenylyltransferase from a recombinant Escherichia coli

Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat, contributing to interest in understanding the mechanisms of resistance. One important mechanism of streptomycin modification is through ATP dependent O-adenylation, catalyzed by streptomycin adenylyltransferase (SMATase). The aim of this study was to purify the recombinant SMATase by Ni(2+)-IDA-His bind resin column chromatography. Thioredoxin-His6-tagged SMATase fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The purified fusion protein showed a single band on SDS-PAGE corresponding to 49 kDa. The recovery of fusion protein was 47% with ninefold purification. The fusion system provided a single step, easy and very rapid purification of SMATase and is suitable for obtaining a highly purified functional protein of interest. The fusion does not affect the functionality of the protein.     

Bacterial expression and characterization of the ligand-binding domain of the vitamin D receptor

The ligand-binding domain of the rat vitamin D receptor (amino acids 115-423) was expressed as an amino-terminal His-tagged protein in a bacterial expression system and purified over Ni-nitrilotriacetic acid resin and a Mono S column. The purified protein bound its ligand, 1,25-dihydroxyvitamin D3, with high affinity, similar to that of the full-length protein. Saturation of the protein with ligand quenched 90% of the tryptophan fluorescence, consistent with the purified protein being uniformly able to bind ligand. Addition of ligand produced no change in the tryptophan fluorescence lifetime, suggesting static quenching as the mechanism of fluorescence decrease. The near-UV circular dichroism spectrum showed a large increase in signal following the addition of ligand, consistent with a change in the environment of aromatic amino acid side chains. The far-UV circular dichroism spectrum was consistent with a protein of high alpha-helical content. Sedimentation equilibrium experiments demonstrated that the protein formed higher-order complexes, and the distribution of the protein among these complexes was significantly shifted by addition of ligand.     

A sensitive method for the assay of guanylate cyclase activity.

A method for the assay of guanylate cyclase is described utilizing alpha-[32P]-GTP as substrate for the enzyme reaction. 100-150 microgram of enzyme protein is incubated in a 15.6 mM Tris-HCl buffer incubation mixture, pH 7.6. The reaction is stopped by the addition of EDTA. The [32P]-cyclic GMP formed is separated by a two-step column chromatography on Dowex 50W-X4 ion-exchange resin and neutral alumina. The recovery for cyclic GMP was about 70%. The blank values ranged from 0.001-0.003% of the added alpha-[32P]-GTP which had been purified by Dowex 50W-X4 column chromatography. This method was employed for the assay of guanylate cyclase activities in different tissues.     

Yeast RNA polymerase III: A zinc metalloenzyme

Atomic absorption spectroscopy has been used to demonstrate that zinc is associated with yeast RNA polymerase III. The enzyme purified by DNA-Sepharose chromatography gives a single predominant protein band in polyacrylamide gel electrophoresis and contains 0.7 gram-atoms of zinc per 100,000 grams of protein. The zinc is tightly associated with the enzyme and cannot be removed by passing the protein through a column of Chelex-100 resin under conditions where free zinc is quantitatively removed. Inhibition by the chelating agent 1,10-phenanthroline demonstrates that the zinc is essential to the catalytic process. The enzyme is inhibited 50% at 0.1 mM and 100% at 1 mM 1,10-phenanthroline.