Expression and purification of the SsbB protein from Streptococcus pneumoniae

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.     

IL-1018-57-PE40高效表达、纯化及细胞活性之研究

以IL-10的功能短肽(40肽,即IL-10第18号至57号氨基酸)为导向部分与PE40(绿脓杆菌外毒素除去受体结合区后的剩余部分)融合分别构建了IL-101857PE40的胞质和胞周质表达质粒,其中,IL101857PE40在Rosettablue(DE3)中以高效胞质可溶形式表达,在BL21(DE3)pLysS中以胞周质分泌形式表达;表达宿主菌Rosettablue(DE3)超声波破碎后,依次通过硫酸铵盐析、疏水层析、铜离子亲和层析、阴离子交换层析纯化后,得96%重组毒素纯品;细胞活性实验、细胞ELISA和荧光标记实验表明,构建的IL101857PE40符合免疫毒素的作用机理。因此,该实验为PE免疫毒素的规模制备和纯化做了一定的有益的探索。     

Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli

Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.     

Expression and purification of soluble non-fusion vasostatin in Escherichia coli

Vasostatin has previously been expressed in fused form or in inclusion body form in Escherichia coli. Here the protein was expressed in soluble non-fusion form in BL21(DE3)pLysS by IPTG induction. The expression level of vasostatin was about 15% of the total cellular protein. The expressed vasostatin was purified and its biological activity was investigated by an endothelial cell proliferation assay.     

IL-1023-57-PE40分泌表达的初步研究

将IL-1023-57-PE40基因与pelB信号肽融合置于pET-20b构建分泌表达质粒pET-20b-IL-1023-57-PE40,然后将pET-20b-IL-1023-57-PE40分别转化至BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3),E·coliK12TB1,ER2566中。无论是在37℃或是在26℃,亦或在培养基中添加葡萄糖的情况下,IPTG诱导后,IL-1023-57-PE40蛋白只在BL21(DE3)pLysS菌中以可溶分泌形式表达,其中以37℃时培养基中不添加葡萄糖表达量为最高,占菌体蛋白总量的15%,说明蛋白的分泌表达与菌种的选择有关。表达产物经免疫印记检测可被抗PE40的特异抗体识别。通过质粒稳定性实验证明,pET-20b-IL-1023-57-PE40在BL21(DE3)中不稳定,导致蛋白的不表达,在Rosetta(DE3)BL21,E·coliK12TB1,ER2566中稳定但不表达,因此,以Rosetta(DE3)BL21为例,通过SDS-PAGE、DNAStar和ANThewin蛋白分析软件对本室构建的几种PE重组毒素进行比较分析,我们发现:并不是所有PE重组毒素融合信号肽序列后,就能分泌表达,PE重组毒素分泌表达还可能与导向部分的性质有关。     

Expression of ornithine decarboxylase of Coccidioides immitis in three Escherichia coli strains carrying the lambda DE3 lysogen and an E. coli EWH319 strain odc − null mutant

Ornithine decarboxylase from respiratory fungal pathogen, Coccidioides immitis, cloned in the pETCiODC plasmid under control of T7lac promoter, was produced in E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3) and EWH319 transformant strains. E. coli BL21(DE3)pLysS-pETCiODC expressed the highest specific activity of ODC, suggesting that this strain could be successfully used for protein structure and drug testing studies.     

Cloning,expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.     

花生△12脂肪酸脱氢酶基因高效表达载体构建与原核表达

花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△^12脂肪酸脱氢酶（FAD2）是亚油酸合成的关键酶,催化油酸（18：1）在△^12位上脱氢生成亚油酸（18：2）,但由于△^12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△^12脂肪酸脱氢酶基因（GenBank接受号为AY1006）构建高效表达载体,把花生△^12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET／HO-A融合表达载体,并转化到大肠杆菌表达菌BL21（DE3）pLysS中,在IPTG诱导下,pRSET／HO-A融合表达载体在BL21（DE3）pLysS菌株中高效表达了△^12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱（GC）和气相色谱,质谱（GC-MS）分析表明,所编码的酶具有△^12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11．8％。花生△^12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。     

在大肠杆菌中表达有活性的人STK11蛋白

STK11蛋白(serine/threonine kinase11)是近年来发现的具有多种重要功能的蛋白,可参与调控细胞周期、p53介导的细胞凋亡、ras诱导的细胞转化、细胞极化等多种生物学过程。利用大肠杆菌高效表达有活性的人STK11蛋白,可为其结构和功能的深入研究打下良好基础。利用本室克隆的人STK11 cDNA和原核表达载体pET-44a( )构建带有Nus融合标签的诱导型表达载体pET-Nus-STK11,在不同的大肠杆菌宿主中诱导表达。SDS-PAGE和Western blot检测表明,在BL21(DE3)宿主中表达的融合蛋白主要以包涵体形式存在,占菌体总蛋白的8.9%;在Rosetta-gami(DE3)pLysS宿主中主要表达为可溶性蛋白,占菌体总蛋白的16.7%。而经纯化和包涵体蛋白复性处理后,以Chariot介导重组融合蛋白进入人肝癌细胞SMMC-7721检测其对细胞生长和细胞周期的影响。与对照组相比,BL21(DE3)中表达的Nus-STK11蛋白几乎无抑制活性;而Rosetta-gami(DE3)pLysS中表达的Nus-STK11蛋白可以显著抑制SMMC-7721细胞的生长,抑制率达47.05%,并导致细胞周期的G0/G1期阻滞,证实表达的重组融合蛋白具有明显的生物学活性。上述结果为在大肠杆菌中成功表达有活性的重组STK11蛋白的首次报道。     

抗CEA免疫毒素的表达、纯化、复性与生物学活性的研究

以抗癌胚抗原（Carcinoembryonic antigen, CEA）单链抗体与假单胞菌外毒素（Pseudomonas exotoxin A, PEA）的截短和修饰形式PE38/KDEL构建重组免疫毒素CEA/PE38/KDEL,并在大肠杆菌菌株BL21(DE3)-star中表达。采用镍离子螯合层析法纯化变性的包涵体样品,并用连续梯度透析的方法对纯化后的包涵体进行复性。采用流式细胞术鉴定复性产物与靶细胞的结合活性,结果表明免疫毒素CEA/PE38/KDEL具有与靶细胞特异性结合的活性。以MTT法检测免疫毒素对肿瘤细胞的体外杀伤活性,结果表明该免疫毒素对SW1116和CNE_2细胞具有特异性杀伤活性。证明了经包涵体复性的抗CEA免疫毒素CEA/PE38/KDEL对表达CEA抗原的肿瘤细胞具有良好的结合和杀伤活性。     

Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.     

Construction and expression of novel immunotoxin cpIL-4(13D)-PE38KDEL with increased activity

Interleukin-4 receptors (IL-4Rs) are expressed on a wide variety of human cancer cells, and therefore it may be a good option to treat IL-4R-bearing tumors with IL-4-fusing immunotoxins. In this study, the gene encoding human interleukin-4 mutein cpIL-4(13D) was obtained through overlapping polymerase chain reaction. A chimeric immunotoxin was constructed by genetically fusing the mutein cpIL-4(13D) to a modified version of Pseudomonas exotoxin A (PE38KDEL) and was expressed in Escherichia coli AD494 (DE3). The expression level of the fusion protein was about 30% of the total bacterial protein assessed by SDS-PAGE analysis. After purification by affinity chromatography and anion exchange chromatography, the chimeric protein was tested for its cytotoxicity. Our data show that cpIL-4(13D)-PE38KDEL has improved cytotoxicity on IL-4R-bearing tumor cells in comparison with other IL-4-fusing immunotoxins and might be useful in treating tumors with a large number of IL-4Rs.     

Specificity of DNA binding activity of the Bacillus subtilis catabolite control protein CcpA.

CcpA was purified from Escherichia coli BL21 (lambda DE3)/pLysS carrying plasmid pTSC5, which was constructed by inserting the ccpA gene into the polycloning site of pGEM4. The purified protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mass of 38 kDa but was eluted from a calibrated Bio-Gel P-100 column with an apparent mass of 75 kDa. Western blot (immunoblot) analysis revealed the presence of CcpA in E. coli BL21 (lambda DE3)/pLysS/pTSC5, which carries ccpA, and in wild-type Bacillus subtilis 168 but not in E. coli BL21 (lambda DE3)/pLysS/pGEM4 or in B. subtilis WLN-29, in which ccpA is inactivated by transposon Tn917 insertion. Purified CcpA bound to DNA containing amyO and retarded its mobility in electrophoretic mobility shift analysis. Complete retardation of the DNA required 75 ng of CcpA per assay. In DNase protection analysis, CcpA bound to DNA containing amyO and protected a region spanning amyO when either DNA strand was labeled. Mutant forms of amyO not effective in catabolite repression were not retarded by CcpA.     

凋亡素融合蛋白的可溶性表达及活性分析

目的:构建PET-28a-SPA原核表达载体,在大肠杆菌BL21(DE3)中实现其高效可溶性表达,测定对肿瘤细胞的凋亡效果。方法:本实验在获得凋亡蛋白融合基因的基础上,成功地构建了重组表达质粒PET-28a-SPA,将阳性重组质粒转化表达受体菌BL21(DE3)感受态细胞中,经IPTG诱导表达,表达产物经聚丙烯酰胺凝胶电泳检测和Western blot检测,并采用MTT法检测其对肿瘤细胞的增殖抑制。结果:表达产物经聚丙烯酰胺凝胶电泳检测,凋亡蛋白融合基因获得高效表达,软件分析表明表达蛋白占菌体蛋白20%左右。上清表达量约为10%。上清蛋白经纯化后,Western blot结果显示,利用凋亡蛋白单克隆抗体可以很好地和所表达的蛋白带特异性结合,并且对A549肺癌细胞及Hela细胞具有一定的凋亡作用。结论:所获凋亡蛋白以高效胞质可溶形式表达,为其研制有效的肿瘤免疫治疗靶向药物提供一定的基础。     

家蝇溶菌酶MDLZM2基因的克隆、序列分析及原核表达

对家蝇溶菌酶(Musca domestica lysozyme,MDLZM2)基因进行克隆、序列分析,构建原核表达载体并在大肠杆菌中表达。从Gen Bank家蝇基因组中筛选获得MDLZM2基因。以该基因的序列设计引物,进行PCR扩增,测序分析获得该基因完整编码序列。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽、二级结构、三级结构和保守结构域等方面进行预测和分析。构建p EASY-E1-MDLZM2重组质粒,转化到大肠杆菌BL21(DE3)p Lys S Chemically Competent Cell中进行诱导表达及纯化。结果表明MDLZM2基因ORF全长552 bp,编码183个氨基酸,理论分子量21.2 k Da;等电点为6.13,具有Lysozyme家族的蛋白保守结构域。成功构建重组原核表达p EASY-E1-MDLZM2并诱导表达、纯化重组蛋白,为进一步研究该蛋白的生物学及免疫学活性奠定了基础。     

人SCYL1-BP1重组蛋白的原核表达及分离纯化鉴定

前期研究结果发现,SCYL1-BP1具有细胞周期调控功能,同时具有肿瘤抑制因子的特性。目的:采用基因工程技术,构建SCYL1-BP1的大肠杆菌重组表达菌株,以获得足够量的高纯度目的蛋白,为后面进行一系列药理学检测及新药安全性测试奠定基础。方法:利用从人胎脑cDNA文库中克隆得到SCYL1-BP1基因克隆为模板,经PCR扩增,通过酶切位点克隆到新型原核表达载体pET-28b-SUMO上,转化大肠杆菌表达菌BL21(DE3)。经IPTG诱导表达,摸索优化表达条件,表达产物经Ni柱进行亲和层析纯化,后再进行SDS-PAGE和Western blot等分析鉴定。结果:成功构建了SCYL1-BP1的原核表达工程菌BL21(DE3)/pET-28b-SUMO-SCYL1BP1。SDS-PAGE和Western blot检验结果表明,诱导表达的融合蛋白His6-SUMO-SCYL1BP1的分子量约为65 kDa,主要以可溶的形式存在,且能被His标签抗体和SCYL1-BP1单克隆抗体特异性识别。结论:原核表达并纯化了人SCYL1-BP1融合蛋白,为其后续功能研究及性质实验奠定基础。     

植原体免疫主导膜蛋白Imp基因原核表达载体构建及表达

旨在构建植原体免疫主导膜蛋白Imp基因原核表达载体,并进行初步表达。以重组克隆质粒pMD18-T-Imp为模板,PCR扩增Imp基因片段。构建表达载体pET-28a(+)-Imp,转化宿主菌E.coliBL21(DE3)。筛选阳性克隆,提取重组质粒作PCR鉴定、酶切鉴定及IPTG诱导表达鉴定。PCR及双酶切结果显示,重组质粒pET-28a(+)-Imp构建成功。经IPTG诱导BL21(pET-28a(+)-Imp)表达约20 kD的蛋白,与预期的携带6×His-Tag的目的蛋白(19.5 kD)大小相符,主要以包涵体形式存在。结果显示,构建的表达载体pET-28a(+)-Imp在E.coliBL21(DE3)中能够达一定量表达,为进一步纯化Imp蛋白奠定基础。     

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies

In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.     

融合基因Mdc-hly的构建及原核表达

构建家蝇天蚕素-人溶菌酶(Mdc-hly)融合基因,实现Mdc-hly基因在大肠杆菌中的表达。通过RT-PCR分别扩增出家蝇天蚕素和人溶菌酶的成熟肽基因序列,再利用Gene-SOEing技术构建融合基因,将融合基因克隆至pET32a表达载体,转化E.coli BL21(DE3),经IPTG诱导得到高效表达,融合蛋白分子量约为38kD。Western blotting杂交证实了表达蛋白的抗原活性。成功构建了融合其因并进行了原核表达,为进一步的生物活性研究打下基础。     

幽门螺杆菌Lpp20融合蛋白的表达、标签切除及鉴定

目的:利用GST融合基因表达系统表达Lpp20-GST融合蛋白,并利用凝血酶切除GST标签.方法:IPTG诱导重组质粒Lpp20/pGEX-4T -1在大肠杆菌BL21 (DE3)中表达,菌体经反复冻融、溶菌酶裂解及超声破菌后,发现Lpp20-GST融合蛋白以部分可溶性的形式表达.采用GST蛋白纯化系统对其纯化,得到Lpp20- GST融合蛋白,再用凝血酶进行GST标签的切除,所得产物进行Western Blot鉴定.结果:高效表达出Lpp20-GST融合蛋白的相对分子质量约4.5kDa,凝血酶成功切除了GST标签,Western Blot证实Lpp20蛋白能被鼠抗Lpp20单克隆抗体识别.结论:成功表达和纯化了重组Lpp20蛋白,为深入研究Lpp20的功能奠定了基础.