蚓激酶基因F238在大肠杆菌中的可溶性表达

应用PCR技术扩增获得蚓激酶成熟肽基因F238,并将其克隆至大肠杆菌质粒pET22b-TrxA中,构建双顺反子表达载体p17rx-F238.重组质粒转化大肠杆菌BL21(DE3),1.0 mmol/LIPTG、30℃条件下诱导6 h,经SDS-PAGE证实蚓激酶成熟肽蛋白获得了高效表达,所表达的蛋白产物相对分子量与预期...     

基于FMDV IRES的双顺反子载体的构建及体外表达分析

利用RT-PCR扩增出口蹄疫病毒小核糖体进入位点（IRES）序列,并定向克隆进pcDNA3.1(+)载体,构建成双顺反子真核表达载体。为了验证该载体是否能够转录出双顺反子mRNA,在IRES起始密码（ATG）下游正确插入增强型的绿色荧光蛋白基因（egfp）,把重组质粒转染BHK-21细胞,培养20～48 h,在紫外显微镜下观察,能够看到典型的绿色荧光,表明载体能够体能够利用FMDV的IRES能够介导非帽依赖性表达外源基因。并通过流式细胞仪,与同样是CMV启动转录egfp的pGFPN1质粒在细胞中的表达的水平进行了比较。该载体的成功构建为体外表达双基因、双顺反子逆转录载体构建以及相关应用奠定基础,并有作为基因疫苗和标记定位基因治疗载体的潜力。     

一种双顺反子表达载体的构建及应用的研究

将表达载体pEC34中的一段寡核苷酸序列,其中包括翻译增强子序列、SD序列、终止码、起始码及两端的限制性内切酶位点,插入GST基因后,构建成双顺反子的表达载体．利用此载体表达了非融合的人骨形成蛋白2A(hBMP2A)和人骨形成蛋白3(hBMP3)C端肽段,将第一顺反子基因（GST基因）切小到原来的1／3时,则位于下游的第二顺反子基因编码的蛋白质在大肠杆菌中的表达量增加一倍。     

大肠杆菌双顺反子表达载体中绿色荧光蛋白表达水平的研究

构建了一系列含有编码硫氧还蛋白(Trx)和绿色荧光蛋白(GFP)基因的双顺反子重组表达质粒,重点考察双顺反子间紧密相连和重迭的间隔序列.诱导表达后,对表达的GFP进行荧光检测,利用GFP的荧光强度来反映蛋白质的表达水平.结果表明各表达载体所表达的GFP的平均荧光强度差距很大,表明双顺反子间的间隔序列对报告基因表达水平有一定的影响,为实现不同功能基因表达的精确调控奠定了基础.     

BALB/c小鼠I-Ad αβ链RFP融合双顺反子表达载体的构建及其在COS-7中的表达

构建了在β链C端融合红色荧光蛋白(RFP)标签的BALB/c小鼠I-Adαβ链真核双顺反子表达载体pRed-IRES-I-Ad,使用LipofectAMINE2000转染COS-7细胞,用激光共聚焦显微镜观察外源蛋白在细胞中的表达与定位.I-Adαβ分子在COS-7细胞中能够以较高的效率表达,并且在COS细胞中能形成聚集状态.与通常的真核翻译帽子结构起始相比,IRES启动真核翻译系统的效率低于前者;与空质粒对比,IRES介导的真核翻译起始,RFP的表达量较低.     

分子伴侣过量表达对蛋白质分泌及可溶性的影响

 通过过量表达大肠杆菌分子伴侣 Sec B和 Gro EL,研究了它们对靶蛋白的分泌及可溶性的影响 .在过量表达 Sec B的宿主菌中 ,周质空间分泌蛋白总量较对照组提高了约 71 % ,GL- 7- ACA酰化酶在周质空间酶的活力较对照组提高了约 1 .5倍 ,碱性磷酸酯酶在周质空间酶的活力较对照组提高了约 54% ;在过量表达 Gro EL的宿主菌中 ,周质分泌蛋白总量较对照组提高了约 52 % ,青霉素 G酰化酶在周质空间酶的活力较对照组提高了约 76% ,鲑鱼降钙素六聚体的可溶性组分的比例由原来的 45%增加到约 90 % ,而 MS2 -人白介素 - 3融合蛋白的包涵体有约 1 5%转变为可溶性组份 .上述结果表明 ,分子伴侣 Sec B和 Gro EL的过量表达促进了靶蛋白的分泌 ,Gro EL增加了靶蛋白的可溶性     

抗真菌肽CGA-N46基因多顺反子表达载体的构建与鉴定

目的:为提高抗真菌肽CGA-N46表达量,对该基因多顺反子表达进行研究.方法:以pEASY-Blunt为克隆栽体,以“pET-30a rbs序列-起始密码子-CGA-N46编码序列-终止密码子”为外源片段,利用同尾酶Nhe I、Spe I和Xba I,构建了含有上述外源片段1、3、5、8拷贝的重组载体pT-CAN46、...     

提高大肠杆菌可溶性重组蛋白表达产率的研究进展

大肠杆菌表达重组蛋白相比真核细胞具有成本低廉、大规模发酵容易、条件易于自动化控制等优点,通过大肠杆菌表达重组蛋白是一种高效、经济的途径,重组蛋白表达量可达到大肠杆菌总蛋白质量的50%。具有正常生化活性的重组蛋白通常为可溶性形式,因而对于以得到活性产物(如抗体、酶等)为目的的研究,通常采用可溶性表达途径。目前已有多种以可溶性重组蛋白为活性物质的治疗性药物经批准上市,但并非所有外源基因均能实现可溶性高表达,因此重组蛋白的可溶性高表达具有重要研究价值。在总结近年提高经大肠杆菌可溶性表达重组蛋白产率研究的基础上,从启动子的选择、SD序列的引入、信号肽的优化、宿主细胞的选择、共表达其他蛋白质,高密度发酵等方面阐释在大肠杆菌中提高可溶性重组蛋白表达产率的方法。     

人TNF-α双顺反子逆转录病毒载体的构建及其在成纤维细胞中的表达

利用脑炎心肌炎病毒的内核糖体进入位点连接人ＴＮＦ－αｃＤＮＡ和选择基因ＮｅｏＲ基因,使ＴＮＦ－α及ＮｅｏＲ基因均受控于病毒ＬＴＲ启动子,将两基因同时转录至同一ｍＲＮＡ,从而构建成人ＴＮＦ－α双顺反子逆转录病毒载体ｐＧＣＥＮ／ＴＮＦ－α．在ＬｉｐｏｆｅｃｔＡＭＩＮＥ介导下将其导入包装细胞ＰＡ３１７,Ｇ４１８筛选得单克隆,病毒滴度为１０６ＣＦＵ／ｍｌ重组病毒分泌的细胞株．经ＰＣＲ证明外源基因已整合至细胞基因组,Ｎｏｒｔｈｅｒｎ印迹显示出单一ＬＲＴ转录本．持续Ｇ４１８筛选能明显促进目的基因ＴＮＦ－α的表达．用重组病毒上清感染小鼠成纤维细胞ＮＩＨ３Ｔ３,Ｇ４１８筛选获得的混合抗性克隆持续高表达ＴＮＦ－α,４０Ｇｙγ线照射后能维持高效表达至７ｄ．实验结果表明,含ＩＲＥＳ的双顺反子逆转录病毒载体将是一个很好的基因转移载体．     

Expression of soluble, biologically active recombinant human endostatin in Escherichia coli

Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in E. coli by employing both co-expression of the molecular chaperones and lower temperature fermentation. Two groups of chaperones Trigger factor and GroEL-GroES (GroEL/ES), DnaK-DnaJ-GrpE and GroEL/ES, were co-expressed, respectively, with rhEndostatin at different temperatures (37, 25, and 16 degrees C). It revealed that low temperature or molecular chaperones alone could enhance the production of active rhEndostatin; meanwhile, combinational employment of low temperature cultivation (16 degrees C) together with co-expression of DnaK-DnaJ-GrpE and GroEL/ES was more effective to prevent aggregation of rhEndostatin. The production of soluble rhEndostatin was about 36 mg/L, and at least 16 mg of rhEndostatin was purified from 1L flask culture. The purified rhEndostatin specifically inhibited the proliferation of endothelial cell-bovine capillary endothelial cell in a dose-dependent manner, and it showed potent anti-angiogenic capability on the chorioallantoic membrane of chick embryo in vivo. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.     

蚓激酶基因的克隆及在毕赤酵母中的表达

以赤子爱胜蚓(Eisenia fetida)体内的总RNA为模板,通过RT-PCR方法扩增含自身信号肽的蚓激酶基因F238,将其克隆到pUCm-T载体上,并进行测序。GenBank登录号为:DQ202401。测序结果表明基因全长为738bp,共编码245个氨基酸,包括7个氨基酸的信号肽序列和238个氨基酸的成熟肽序列。与粉正蚓(Lumbricus rubellus)F-III-2相比,核苷酸与氨基酸序列的同源性均为99%,仅存在2个碱基的差异,导致2个氨基酸的突变。通过生物信息学方法对蛋白质的理化及结构特性进行分析预测,F238的等电点为4.61,含有11个半胱氨酸,形成3个二硫键。蛋白质分子主要由β折叠组成,具有丝氨酸活性中心,属丝氨酸蛋白酶超家族胰蛋白酶类。以重组质粒pUCm-T-F238为模板,通过PCR方法扩增去信号肽的蚓激酶基因F238-m,构建毕赤酵母(Pichia pastoris)表达载体pPIC9-F238-m,将其线性化后用电穿孔法导入酵母宿主菌GS115中。在MM和MD平板上筛选表型,经甲醇诱导后,SDS-PAGE分析显示表达产物的分子量为28kDa左右,纤维平板法测定活力最高可达100U/mL。     

Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian expression vectors

Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5’ of the IRES site influences both expression of the 3’ fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5’ gene of interest sequence (<300 bp), selection of single cells based on the expression profile of the 3’ optogenetic fluorescent marker, and assumptions made during data analysis.     

Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening

Proteins are the main reagents for structural, biomedical, and biotechnological studies; however, some important challenges remain concerning protein solubility and stability. Numerous strategies have been developed, with some success, to mitigate these challenges, but a universal strategy is still elusive. Currently, researchers face a plethora of alternatives for the expression of the target protein, which generates a great diversity of conditions to be evaluated. Among these, different promoter strength, diverse expression host and constructs, or special culture conditions have an important role in protein solubility. With the arrival of automated high-throughput screening (HTS) systems, the evaluation of hundreds of different conditions within reasonable cost and time limits is possible. This technology increases the chances to obtain the target protein in a pure, soluble, and stable state. This review focuses on some of the most commonly used strategies for the expression of recombinant proteins in the enterobacterium Escherichia coli, including the use of HTS for the production of soluble proteins.     

人内皮抑素基因的克隆以及在大肠杆菌中的可溶性表达研究

用PCR的方法从人胎肝cDNA文库中得到人内皮抑素基因 ,克隆测序正确后连接到硫氧还蛋白融合表达载体上 ,转化大肠杆菌BL2 1 (DE3)得到表达人内皮抑素的工程菌。用IPTG诱导表达 ,表达量达到全菌蛋白的 64%。经分析硫氧还蛋白可以辅助内皮抑素可溶性表达 ,表达的融合蛋白保持了天然蛋白的免疫学特性。而且表面带有多聚组氨酸的突变的硫氧还蛋白还简化了蛋白纯化的步骤 ,使融合蛋白可以通过固相金属螯和层析 (IMAC)的方法纯化。纯化后的融合蛋白经IgA蛋白酶的切割可得到大小正确的重组人内皮抑素 ,用此方法获得的重组人内皮抑素可以在CAM试验中抑制新生血管的形成。高效可溶型表达内皮抑素的工程菌的构建成功 ,为内皮抑素的生产应用打下了良好的基础。     

Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase Lip-948

The cold-active lipase gene Lip-948, cloned from Antarctic psychrotrophic bacterium Psychrobacter sp. G, was ligated into plasmid pColdI. The recombinant plasmid pColdI+Lip-948 was then transformed into Escherichia coli BL21. SDS-PAGE analysis showed that there was substantive expression of lipase LIP-948 in E. coli with a yield of about 39% of total protein, most of which was present in the inclusion body. The soluble protein LIP-948 only consisted of 1.7% of total LIP-948 with a specific activity of 66.51U/mg. Co-expression of molecular chaperones with the pColdI+Lip-948 were also carried out. The results showed that co-expression of different chaperones led to an increase or decrease in the formation of soluble LIP-948 in varying degrees. Co-expression of pColdI+Lip-948 with chaperone pTf16 and pGro7 decreased the amount of soluble LIP-948, while the soluble expression was enhanced when pColdI+Lip-948 was co-expressed with "chaperone team" plasmids (pKJE7, pG-Tf2, pG-KJE8), respectively. LIP-948 was most efficiently expressed in soluble form when it was co-expressed with pG-KJE8, which was up to 19.8% of intracellular soluble proteins and with a specific activity of 108.77U/mg. The soluble LIP-948 was purified with amylase affinity chromatography and its enzymatic characters were studied. The optimal temperature and pH of LIP-948 was 35°C and 8, respectively. The activity of LIP-948 dropped dramatically after incubation at 50°C for 15min and was enhanced by Sr(2+), Ca(2+). It preferentially hydrolyzed 4-nitrophenyl esters with the shorter carbon chain.     

PHD2基因原核表达载体的构建及其在大肠埃希菌中的表达

目的构建PHD2基因原核表达载体pET-43.1b（＋）-PHD2,实现Nus-PHD2融合蛋白在大肠埃希菌中的可溶性表达。方法用SacⅠ酶切pET-43.1b（＋）制备线性化载体,设计与线性化载体两端具有至少15个同源序列的特异性引物,以真核重组质粒pCMV6-Entry-EGLN1为模板,PCR法扩增PHD2目的基因。采用In-Fusion技术构建原核表达载体pET-43.1b（＋）-PHD2,并将其导入大肠埃希菌BL21（DE3）中诱导表达。用SDS-PAGE和Western blot分析并鉴定表达出的融合蛋白。用Ni-NTA亲和层析法纯化目的蛋白。结果成功构建了PHD2原核表达载体;SDS-PAGE结果显示融合蛋白以可溶性形式表达;Western blot鉴定表明融合蛋白可以与PHD2单克隆抗体特异性结合。结论实现了Nus-PHD2融合蛋白在大肠埃希菌中的可溶性表达,为PHD2生物学功能的研究奠定了基础。     

蛇毒纤溶酶Alfimeprase在大肠杆菌中的可溶表达和纯化

Alfimeprase是Fibrolase的突变体,是一种蛇毒纤溶酶,有纤溶活性而无出血性。根据Alfimeprase的氨基酸序列和大肠杆菌密码子偏爱性,利用PCR的方法合成Alfimeprase DNA序列,分别融合在NusA和MBP的C端,与分子伴侣FkpA在大肠杆菌Origami B(DE3)中共表达,融合蛋白NusA/Alfimeprase以部分可溶的形式存在,可溶部分占上清总蛋白的25%左右,通过镍柱亲合层析纯化和肠激酶切割得到具有纤溶活性的重组蛋白Alfimeprase。本研究是首次报道在大肠杆菌中可溶表达Alfimeprase,为以后深入研究其功能及应用奠定了基础。