Expression and partial purification of human prolactin in Escherichia coli

1. Human prolactin has been expressed in Escherichia coli. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E. coli. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.     

Purification and biological characterization of bacterially expressed recombinant buffalo prolactin

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22?mg/L from 100?mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT BUFFALO PROLACTIN

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

A bacterial cell that synthesizes a protein containing the antigenic determinants of rat prolactin.

Bacterial minicells containing three different recombinant plasmids with rat prolactin cDNA sequences inserted at the Pst I site of pBR322 via the poly(dG):poly(dC) joining technique were examined for the expression of rat prolactin antigenic determinants. The three prolactin coding sequences were in the same orientation as the coding sequence of the ampicillin-resistance gene of pBR322. The presence of each of the three recombinant plasmids induced some prolactin synthesis by the bacteria as measured by immunoprecipitation with anti-prolactin antisera. About 10% of the protein synthesized from one of the plasmids, prl 3, precipitated with the antisera. These prolactin antigenic determinants were part of a larger fused protein.     

白鹅催乳素基因的克隆及诱导表达条件的优化

摘要: 运用RT-PCR方法, 从白鹅脑垂体总RNA中扩增得到了催乳素(Prolactin, PRL)基因编码区序列cDNA, 并将其克隆到pMD18-T载体上。DNA序列分析表明, PRL cDNA包括终止密码子在内的长度为690 bp,编码230个氨基酸残基的蛋白质, 与皖西白鹅的有所差异, 二者碱基同源性在99.57%, 氨基酸同源性达99.56%。将PRL基因编码区序列cDNA定向克隆到表达载体pET-32a (+)中, 构建表达质粒pET-32a(+)-PRL。该质粒的BL21 (DE3)转化菌在IPTG的诱导下可表达PRL基因融合蛋白, IPTG终浓度1 mmol/L, 37℃, 诱导4 h表达量最高, 表达量约占菌体总蛋白的28.96%。     

Production of a polyclonal antibody against recombinant goldfish prolactin and demonstration of its usefulness in a non-competitive antigen-capture ELISA

The cDNA encoding the goldfish (Carassius auratus) prolactin was expressed in Escherichia coli using the pRSETA expression vector. The recombinant goldfish prolactin (gfPRL) produced was a fusion protein containing a hexahistidyl sequence, which facilitated its purification on a Ni²⁺ column. The fusion protein was overexpressed in the bacteria as inclusion bodies and was successfully purified under denaturing conditions by one-step affinity chromatography. Repeated immunization of rabbits against the purified recombinant gfPRL allowed the production of a high-titer polyclonal antiserum. The IgG fraction of the antiserum was isolated on an immobilized Protein A–agarose column. The antibody recognized recombinant gfPRL, but not recombinant goldfish growth hormone (gfGH) or goldfish somatolactin (gfSL) on Western analyses. The purified antibody was able to recognize gfPRL, but not gfGH or gfSL, in a non-competitive antigen-capture ELISA. The assay was applied in monitoring the purification of native PRL from goldfish pituitaries.     

人源抗TNF—α抗体Fab基因的单链化及其表达和纯化

构建人源抗TNF－α单链抗体基因,并尝试其在E.coli中的表达和纯化,采用人工接头,按VH－linkerVL的结构将人源抗TNF－a的VH,VL基因拼接成单链抗体（ScFv)基因,并将构建好的ScFv基因插入表达载体pQE30,转染E.coli M15,以IPTG诱导表达,Ni-NTA树脂纯化表达产物,构建的ScFv基因长723bp, 列分析表明,该序列拼接正确,SDS－PAGE显示,用重组的pQE30转化的M15菌经诱导后,有相对分子质量（M）约为52000的外源蛋白表达,Ni-NTA树脂纯化的表达产物纯度大于90％,因此,成功地构建了人源抗TNFa的ScFv基因,并在E.coli DH5a中表达和纯化的该基因的产物。     

人精氨酸酶在毕赤酵母的高效表达、纯化和活性研究

目的：人精氨酸酶（Arginase, Arg）的基因arg在毕赤酵母高效分泌表达,建立相应纯化工艺路线,研究重组人精氨酸酶的活性。方法：将人精氨酸酶基因arg按正确的阅读框架插入到毕赤酵母表达载体pPIC9α信号肽基因后,构建得到重组毕赤酵母表达质粒。转化毕赤酵母GS115筛选高表达菌株。结果：成功构建了酵母表达载体pPIC-Arg,转化毕赤酵母GS115后筛选到分泌表达目的蛋白Arg的菌株,目标蛋白可以分泌到培养基中。经过膜过滤和凝胶过滤层析对培养基上清进行纯化,即可获得纯度达到95%的活性产物。活性测定表明,纯化的Arg比活性为310 IU/mg。结论：成功构建了Arg的毕赤酵母高效表达菌种,建立了目标物质的分离纯化工艺。     

自噬相关基因Atg5的原核表达及多克隆抗体制备

目的：克隆自噬相关基因Atg5,在大肠杆菌中重组表达后制备抗Atg5多克隆抗体。方法：用RT-PCR方法从RAW264．7细胞基因组中克隆Atg5基因,连接至pQE80L原核表达载体后转化大肠杆菌DH50α进行诱导表达,SDS-PAGE及Westernblot鉴定表达蛋白;目的蛋白纯化后,以100μg／kg纯化的蛋白免疫新西兰兔,制备抗Atg5多克隆抗体;提取RAW264．7细胞总蛋白,以制备的抗Atg5多抗进行Westernblot反应,检测多抗的生物学活性。结果：克隆了Atg5基因,在大肠杆菌中表达了重组Atg5,SDS-PAGE分析显示表达产物相对分子质量与预期值一致,Western blot结果证明该产物具有较高的生物学活性,纯化蛋白免疫动物后制备了抗Atg5多克隆抗体。结论：在大肠杆菌中表达了重组Atg5,制备了抗Atg5多克隆抗体,为自噬的检测和研究提供了工具。     

Differential expression of the Ebola virus GP(1,2) protein and its fragments in E. coli

Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP(1,2) gene and subfragments of the GP(1) gene were cloned in a bacterial expression vector as a C-terminal His(6) fusion protein. Surprisingly, the full length EBOV GP(1,2) gene could not be expressed in Escherichia coli. The subfragments of GP(1) were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP(1,2), GP(1) subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP(1) subfragment D protein was expressed exclusively as an insoluble approximately 25 kDa His(6) fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP(1) subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP(1,2) protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.     

MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.     

细极链格孢菌peaT2基因在毕赤酵母中的表达及蛋白功能确定

从细极链格孢菌表达文库获得阳性克隆子,序列分析表明,克隆的DNA片段中含有完整的开放阅读框架,将该基因命名为peaT2(GenBank登录号为EF212880)。用PCR法扩增peaT2基因的编码序列并亚克隆到毕赤酵母表达系统的表达载体pPIC9K上,得到重组质粒pPIC9K/peaT2。重组质粒经SacⅠ线性化后用电穿孔法导入到毕赤酵母(Pichia pastoris)GS115中,采用MD、G418-YPD平板和PCR法筛选Mut+表型,获得了分泌表达的重组毕赤酵母。随机挑取一菌株作为表达菌,用甲醇诱导PeaT2蛋白表达。SDS-PAGE及Western blot检测结果均表明PeaT2在毕赤酵母中成功地分泌表达。用peaT2基因的表达蛋白处理小麦种子,生物测定表明,表达蛋白能明显促进小麦的生长,具有蛋白激发子作用。     

嗜热古菌S-腺苷甲硫氨酸合成酶的序列分析、基因克隆与异源表达

将Methanopyrus sp.SNP6进行功能基因组测序,获得S-腺苷甲硫氨酸合成酶基因(sam)序列,并进行序列分析。将sam基因扩增后连接至表达质粒p ET-28b(+),转化E.coli BL21(DE3),获得重组菌后进行诱导表达。生物信息学分析表明,sam基因编码蛋白的理论分子量为44 086.4Da,三级结构为同源四聚体,与其他相关古菌来源的S-腺苷甲硫氨酸合成酶的蛋白序列较保守。实验结果显示,构建的重组表达质粒p ET28b(+)-sam可在E.coli BL21(DE3)宿主菌中高水平表达。重组蛋白的分子量与预期值基本一致,部分为胞内可溶性表达,另一部分以包涵体形式存在。本研究首次实现了Methanopyrus sp.SNP6菌株S-腺苷甲硫氨酸合成酶的异源表达,为后期的蛋白纯化、酶学性质研究和酶促转化法生产S-腺苷甲硫氨酸奠定了理论基础。     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

拟南芥中柯浩体蛋白Atcoilin的克隆、表达及纯化

旨在制备柯浩体的标志蛋白——Atcoilin蛋白,利用pET-28a与目的基因构建重组表达质粒,经DNA测序证实插入序列与设计完全一致后,将重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,产物用SDS-PAGE及Western blotting分析鉴定。通过分别改变IPTG的浓度、培养时间、培养温度等来优化Atcoilin蛋白的表达条件。表达出的重组蛋白经过镍柱、分子筛进行纯化。结果显示,原核表达载体pET28a-At1g13030成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相应的重组蛋白经Western blotting鉴定正确。在IPTG浓度为0.7 mmol/L,18℃培养20 h的条件下,目的蛋白表达量最高。经过SDS-PAGE分析鉴定,过镍柱、分子筛后得到的重组蛋白纯度较高。     

抗菌肽CM4与人可溶性B淋巴细胞因子hsBAFF在大肠杆菌中的融合表达

为进一步探讨抗菌肽CM4的原核表达及其生物学功能,本实验研究了抗菌肽CM4与人可溶性B淋巴细胞刺激因子hsBAFF的融合表达及抗菌肽CM4的生物学活性。运用PCR把B淋巴细胞因子hsBAFF和家蚕抗菌肽CM4进行基因融合,构建了融合表达载体pET28a (+)/CM4-hsBAFF,并在大肠杆菌中获得高可溶性表达的融合靶蛋白,且存在于超声破碎后的上清,经分子筛Sephadex G-75纯化后的重组融合蛋白用SDS-PAGE和Western blot分析鉴定.SDS-PAGE分析表明：可以通过分子筛一步纯化得到融合蛋白,该重组融合蛋白的分子量约22.0 KDa。Western blot结果显示该重组蛋白能与鼠抗人hsBAFF的抗体发生特异性反应.运用基因工程的方法获得CM4-hsBAFF重组融合蛋白,并具有很好的抑菌生物学活性。     

His-FemA融合蛋白表达载体的构建及其在原核细胞中的表达

目的 构建His标签的金黄色葡萄球菌甲氧西林耐药相关蛋白FemA的融合蛋白表达载体,并在大肠埃希菌中表达,为进一步研究femA基因的生物学功能和临床应用奠定基础.方法 根据GenBank中金黄色葡萄球菌femA基因序列,利用Primer Premier 5.0设计PCR引物,并在引物的5'加入BamHI及SalI酶切位点;以金黄色葡萄球菌基因组DNA为模版,PCR扩增出femA基因片段.将目的DNA片段及质粒pQE30分别进行双酶切、连接并转化大肠埃希菌DH5α;阳性克隆以PCR、双酶切及测序进行鉴定.将鉴定正确的pQE30-femA重组质粒转化大肠埃希菌JM109,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His-femA融合蛋白;采用SDS-PAGE及Western blot分析对表达蛋白进行验证.结果 经PCR、双酶切签定及序列测定,证实重组质粒pQE30-femA构建成功;重组质粒pQE30-femA转化大肠埃希菌JM109经IPTG诱导后,SDS-PAGE和Western blot分析显示表达出53 kD目的蛋白;经Bandscan软件分析,目的蛋白质在4h的表达量占细胞总蛋白的27.5％.结论 成功构建了His-FemA原核表达载体(pQE30-femA),并在大肠埃希菌中高效表达.