A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain

A new high-yield yeast expression/secretion system has beenadapted for the plant thiol endoprotease papain. The propapaingene, obtained from Carica papaya fruit, is expressed in theyeast Saccharomyces cerevisiae. The gene was cloned into a FLAGepitope-tagging expression vector downstream of the yeast alphamating factor (-factor) secretion signal sequence. Expressionof the heterologous propapain in yeast is controlled by theglucose-repressible alcohol dehydrogenase isoenzyme II promoter(ADH2). Glycosylated FLAG-tagged propapain is secreted by asocalled ‘super secretor’ strain, pmr1 (ssc1), intothe culture supernatant where it accumulates to {small tilde}1.7mg/1. The proregion contains three consensus N-linked glycosylationsites, whereas there are only two such sites in previously reportedcDNA sequences. Removal of this third N-linked glycosylationsite results in a drastic reduction in the level of proteaseactivity present in the culture supernatant. Two different typesof affinity chromatography were used to purify either propapainor papain. The propapain precursor is autoproteolytically activatedto mature papain (M_r = 24 kDa) using conditions reported previously.The kinetic parameters obtained agree well with the literaturevalues. The yields of active papain are 10-fold higher thanthose previously reported for propapain in other yeast or bacterialexpression systems. This, together with the ease with whichmutant proteins can be made, makes this yeast advantageous fora structure–function analysis of recombinant wild-typeand mutant papain, and possibly for other related cysteine proteasesas well.     

Gene expression response to misfolded protein as a screen for soluble recombinant protein

Proper protein folding is key to producing recombinant proteinsfor structure determination. We have examined the effect ofmisfolded recombinant protein on gene expression in Escherichiacoli. Comparison of expression patterns indicates a unique setof genes responding to translational misfolding. The responseis in part analogous to heat shock and suggests a translationalcomponent to the regulation. We have further utilized the expressioninformation to generate reporters responsive to protein misfolding.These reporters were used to identify properly folded recombinantproteins and to create soluble domains of insoluble proteinsfor structural studies.     

不同信号肽对重组B群脑膜炎奈瑟菌H因子结合蛋白表达的影响

目的探讨不同信号肽对重组B群脑膜炎奈瑟菌H因子结合蛋白(factor H-binding protein,f HBP)表达的影响。方法运用PCR方法将B群脑膜炎奈瑟菌f HBP原始信号肽分别替换为大肠埃希菌主要外膜脂蛋白(major outer membrane lipoprotein,MLP)、脂蛋白-28(lipoprotein-28,LP)和流感嗜血杆菌(Haemophilus influenzae)P4外膜蛋白信号肽,并将其分别连接至p ET-30a表达载体,转化入大肠埃希菌感受态细胞,IPTG诱导表达,SDS-PAGE鉴定蛋白表达水平。结果替换了P4、LP和MLP信号肽的重组B群脑膜炎奈瑟菌f HBP表达量较带原始信号肽的B群脑膜炎奈瑟菌f HBP相比,分别提高了2.68、1.94和2.91倍。结论替换MLP信号肽对重组B群脑膜炎奈瑟菌f HBP蛋白表达的影响最大,为提高B群脑膜炎奈瑟菌f HBP表达量的研究工作提供了新思路。     

A transient expression vector for recombinant protein production in Chinese hamster ovary cells

An expression vector was specifically designed for use in Chinese hamster ovary (CHO) cells to enhance the level of protein production in a transient expression system. Two key components that can increase protein production transiently are the promoter used to drive recombinant gene expression and the template copy number. In this study the modified and metal‐inducible metallothionein (M2.6) promoter was shown to be superior to the human cytomegalovirus (CMV) and to the simian virus SV40 promoters. Plasmid replication was achieved using the Polyoma (Py) virus origin of replication (PyOri) and the Py Large T antigen (PyLT). An expression vector containing Py elements was shown to replicate extensively in CHO cells. The combination of the metal‐inducible M2.6 promoter and episomal replication of the expression vector, named pPyOriLT resulted in elevated levels of transgene expression following transient transfection of CHO cells Copyright © 2005 Society of Chemical Industry     

重组EB病毒Zta蛋白的原核表达及纯化

目的原核表达重组EB病毒(Epstein-Barr virus,EBV)Zta蛋白,并进行纯化。方法 PCR扩增EBV B95-8株BZLF1n氨基端基因,分别插入pThioHisA和pcDNA3.1载体中,构建重组表达质粒pThioHisA-BZLF1n和pcDNA3.1-BZLF1n。用pcDNA3.1-BZLF1n质粒免疫ICR小鼠,制备抗血清。将质粒pThioHisA-BZLF1n转化大肠杆菌BL21(DE3),IPTG诱导表达,优化诱导表达条件。表达产物经亲和层析后,用肠激酶切割,再经离子交换层析,纯化产物进行SDS-PAGE和Western blot分析。结果重组表达质粒经双酶切和测序证明构建正确。表达的重组蛋白相对分子质量约为32 000;IPTG终浓度为0.1 mmol/L,37℃诱导6 h,目的蛋白的表达量最高,占菌体总蛋白的30%以上;重组蛋白以包涵体和可溶性两种形式表达。纯化的重组蛋白相对分子质量约为18 000,纯度大于95%,可与制备的小鼠抗血清发生特异性反应。结论在大肠杆菌中高效表达了重组EBV Zta蛋白,纯化的重组蛋白纯度较高,特异性良好,为EBV相关疾病的早期诊断筛查奠定了基础。     

Wistar大鼠热休克蛋白70基因重组腺病毒质粒的构建及病毒制备

目的构建Wistar大鼠热休克蛋白(Heat shock protein 70,HSP70)基因重组腺病毒质粒,并制备重组腺病毒。方法利用RT-PCR技术从Wistar大鼠脾脏组织中扩增HSP70基因序列,并克隆至pMD18-T载体中,测序鉴定后,将克隆的HSP70基因编码阅读框亚克隆至穿梭载体pShuttle-CMV中,并利用Ⅰ-CeuⅠ与Ⅰ-SceⅠ将重组穿梭质粒上的HSP70基因的表达框切下,连接到携带腺病毒骨架载体pAdxsi上。重组腺病毒质粒经PacⅠ酶切线性化后,转染至293(R)细胞进行病毒的包装,获得重组腺病毒pAd-CMV-HSP70,收集病毒上清,感染BRL细胞,Western blot检测BRL细胞中HSP70的表达。病毒颗粒经氯化铯密度梯度离心纯化后,检测重组腺病毒纯度、颗粒滴度及感染性滴度。结果克隆质粒、穿梭质粒经PCR及酶切鉴定,均可见2 269 bp的目的基因条带,测序结果与GenBank登录的序列一致;XhoⅠ酶切结果显示,HSP70基因已正确克隆至腺病毒质粒中;重组腺病毒感染BRL细胞后,细胞中HSP70的表达量显著升高(P<0.01);重组腺病毒纯化后纯度为1.29,颗粒滴度为5.31×1012VP/ml,感染性滴度达1×1011pfu/ml。结论已成功构建Wistar大鼠HSP70基因重组腺病毒质粒,并制备出高滴度的重组腺病毒颗粒,为进一步研究HSP70的生物学活性及作用机制奠定了基础。     

重组人骨形态发生蛋白7成熟蛋白在悬浮CHO-S细胞中的稳定表达

目的在悬浮CHO-S细胞中稳定表达重组人骨形态发生蛋白7(recombinant human bone morphgenetic protein7,rhBMP7)成熟蛋白。方法应用RT-PCR技术从BALB/c乳鼠股骨组织扩增mbmp7基因,克隆至质粒pMD18-T,通过定点突变3个氨基酸编码序列(G266S、S287N、D359E),得到重组克隆质粒pMD18-mbmp7_m3;将mbmp7_m3克隆至表达载体pCHO1. 0,构建重组表达质粒pCHO-mbmp7_m3,转染CHO-S细胞,利用嘌呤霉素和甲氨蝶呤进行两轮加压筛选,有限稀释法筛选单克隆细胞,ELISA法检测rhBMP7成熟蛋白的表达。结果从乳鼠股骨组织扩增的cDNA经测序与GenBank中登录的序列完全一致,点突变后测序与设计完全一致。获得了稳定表达rhBMP7成熟蛋白的单克隆细胞株,最高表达量为202 ng/mL。结论成功利用改造的mbmp7基因在CHO-S细胞中表达了rhBMP7成熟蛋白,为后续该制品工艺开发及产业化奠定了基础。     

A33scFv-green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting

Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.     

新型异亮氨酸双加氧酶及其重组大肠杆菌合成羟基异亮氨酸

异亮氨酸双加氧酶（IDO）可特异性的转化底物L-异亮氨酸（L-Ile）生成4-羟基-L-异亮氨酸（4-HIL）,该产物具有促进胰岛素分泌的功能,可用于抗糖尿病、降胆固醇等。本研究结合了酶标显色和薄层层析（TLC）的方法从自然界中筛到了具有IDO活性的菌株,并将该菌株中的目的基因ido克隆到大肠杆菌中,获得重组表达菌株,并且验证该菌具有IDO的转化功能。本研究优化了转化反应体系和条件,同时通过30℃过夜温育菌体细胞的方法,使该菌株全细胞转化合成4-HIL的产率达到85%以上。     

New expression method and characterization of recombinant human granulocyte colony stimulating factor in a stable protein formulation

Human recombinant granulocyte colony stimulating factor (rhG-CSF) is widely used in hematology and oncology for the treatment of neutropenia, for the restoration of neutrophil production after bone marrow transplantation, for myelodysplastic syndromes, and aplastic anemia. The E. coli expression system is commonly used for fast recombinant production of rhG-CSF at a large scale. We have applied a novel autoinduction method for the batch expression of rhG-CSF to study whether this new system would increase cell mass and target-protein yield compared to conventional E. coli cell culture and induction with isopropyl β-D-thiogalactopyranoside (IPTG). We could demonstrate 3-fold higher culture densities and a 5-fold higher protein yield compared to IPTG induction without the need to monitor cell growth in a shortened 24 h expression procedure. rhG-CSF expressed in autoinduction media was successfully extracted from E. coli inclusion bodies and refolded by dialysis. After size exclusion chromatography (SEC) purification, rhG-CSF showed similar conformation, biological activity and aggregation profile compared to the commercially available biosimilar TEVAgrastim(?) (TEVA Pharma AG). Expression by autoinduction is suggested as a cost- and time-effective method for rhG-CSF production.     

Expression and secretion of a recombinant ricin immunotoxin from murine myeloma cells

Expression plasmids carrying a humanized N901 immuno-globulinheavy chain gene (hN901HC) fused to a gene encoding the nativeB chain of ricin toxin (RTB), hN90 W CRTB, or a sugar bindingmutant of RTB, hN901HC-RTBgly, were constructed. In each case,the fused gene constructions were co-expressed in murine myelomacells (Sp2/0) with the gene for humanized N901 immunoglobulinlight chain to produce the secreted recombinant products hN901-RTBand hN901-RTBgly, respectively. When purified by affinity chromatography,both the hN901-RTB and hN901-RTBgly products were found to havean apparent molecular mass of M_r = 210 000 and to be composedof two hN901 antibody heavy chains each fused to a fulllengthcopy of RTB and two hN901 antibody light chains. In each ofthe recombinant fusions the hN901 antibody moiety retained thefull binding affinity and specificity for its cognate antigen,CD56. Moreover, when mixtures of hN901-RTB and native ricinA chain were incubated in the presence of the antigen-positivetarget cell line SW-2, antigen-specific potentiation of ricinA chain cytotoxicity was observed. It has been demonstratedpreviously that lectin activity of the B chain is essentialfor A chain cytotoxicity, and we conclude that the fused wild-typeB chain was properly folded and maintained lectin activity.These data demonstrate the feasibility of using recombinantricin B chain in an immunotoxin and of using mammalian cellculture for its expression. The use of recombinant hN901-RTBfusion protein to evaluate the contribution of the lectin activityof ricin B chain in the penetration of cell membranes by ricinA chain is proposed.     

Modification of a novel protein product

The production of single cell protein (SCP) on an industrial scale has now been established using the ICI ‘Pruteen’ process. An economic process for the reduction of nucleic acid in the product has been developed: potentially it could be used to produce a protein concentrate suitable for human consumption. Controlled enzymic hydrolysis of this concentrate yields a range of protein products with improved functional properties.     

Frame shuffling: a novel method for in vitro protein evolution

We describe 'frame shuffling', a novel method for preparing artificial protein libraries. With this method, a Y-family DNA polymerase known to introduce frame shift mutations at high rates is utilized to scramble the reading frames of a parental gene. The resultant progeny produce mutant proteins having segmental sequence changes. Such frame-shuffled mutant proteins exhibit physicochemical properties that differ from those of proteins obtained using conventional mutagenesis.     

Engineering trypsin-sensitive sites in a membrane transport protein

This paper describes a systematic procedure for introducing protease- sensitive sites into bacterial integral membrane proteins. Such sites should make it possible to monitor the subcellular localization of individual domains of a topologically complex protein. Escherichia coli lac permease was used as a model. Site-directed mutagenesis, targeted to a particular periplasmic domain, was used to generate insertion derivatives containing a lysine residue in different sequence contexts. Individual mutants were then screened for lactose transport activity and efficient cleavage by trypsin. To facilitate this screen, the mutagenesis was carried out using a gene fusion encoding an easily detected, bifunctional lac permease-galactosidase hybrid. Insertions were identified in the fourth and sixth periplasmic domains (P4 and P6) that were efficiently cleaved in both the hybrid protein and in unfused lac permease. One of the P6 insertion mutants exhibited lactose transport specific activity near that of the wild-type and was shown by sequence analysis to be cleaved at the expected site in the inserted sequence. As part of this analysis, we determined the range of cellular concentrations of lac permease over which lactose uptake was linear. The activity showed a plateau at a relatively low concentration corresponding to approximately five times the wild-type level.      

Fancd2os基因及其编码蛋白的生物信息学分析和表达谱鉴定

目的利用生物信息学手段对Fancd2os基因及其编码蛋白进行结构分析和功能预测,并检测该基因在小鼠组织中的表达。方法利用生物信息学分析软件和技术对该基因及其编码蛋白的同源性、结构定位、理化性质及表达谱等特征进行分析和比对;采用半定量RT-PCR、实时定量PCR及Western blot法分别检测Fancd2os m RNA及其编码蛋白在雄性小鼠不同组织以及不同发育阶段睾丸组织的表达;利用免疫组织化学染色方法检测Fancd2os蛋白在小鼠曲细精管中的细胞定位。结果生物信息学分析表明小鼠Fancd2os蛋白的氨基酸系列与人、大鼠和牛的同源性分别为92.1%、97.8%和93.3%,该蛋白主要定位在细胞浆,二级结构以无规则卷曲和α螺旋为主,无信号肽,含有多个磷酸化位点;EST电子表达谱显示该基因主要在睾丸组织中表达;GEO数据库分析提示Fancd2os基因的表达可能与线粒体Clp P蛋白酶的表达密切相关;MGI Interaction Explorer数据库检索发现Fancd2os基因与95个mi RNA具有相互作用。Fancd2os基因及其编码蛋白在小鼠睾丸组织中表达量高,且具有明显的时序性,以8周龄小鼠表达水平最高;Fancd2os蛋白主要定位于曲细精管精母细胞和圆形精子细胞的胞质中。结论 Fancd2os基因为小鼠睾丸组织高表达基因,其表达水平随着睾丸的发育过程呈上调趋势,结合生物信息学分析结果,该基因可能与睾丸的发育及生精过程相关。     

重组人淋巴细胞活化基因-3蛋白胞外段的真核表达及鉴定

目的真核表达重组人淋巴细胞活化基因-3(lymphocyte activation gene-3,LAG-3)蛋白胞外段,并进行鉴定。方法用植物血球凝集素(phytohaemagg lutinin,PHA)刺激Jurkat细胞,流式细胞术检测Jurkat细胞中LAG-3蛋白的表达;提取Jurkat细胞总mRNA,RT-PCR法扩增人LAG-3蛋白胞外段基因片段,同时在蛋白C-末端引入His标签,将其克隆入载体pcDNA3. 1+,构建重组质粒,转染Expi293F真核细胞,当细胞活率低于50%时收获细胞上清,经镍柱亲合层析纯化。纯化产物进行4%～20%SDS-PAGE、HPLC及Western blot分析,BCA法测定浓度。结果经菌液PCR、双酶切及测序鉴定,表明质粒构建正确。重组表达蛋白的相对分子质量约60 000,纯化后纯度达95%以上,与鼠抗LAG-3单克隆抗体可发生特异性结合,浓度为2. 4 mg/mL。结论成功构建了重组真核表达质粒LAG-3/pcDNA3. 1+,并于Expi293F细胞中表达,纯化获得了纯度较高的LAG-3蛋白,为后期LAG-3蛋白的相关研究及其单抗的制...     

Engineering streptococcal protein G for increased alkaline stability

Most protein-based affinity chromatography media are very sensitivetowards alkaline treatment, which is a preferred method forregeneration and removal of contaminants from the purificationdevices in industrial applications. In a previous study, weconcluded that a simple and straightforward strategy consistingof replacing asparagine residues could improve the stabilitytowards alkaline conditions. In this study, we have shown thepotential of this rationale by stabilizing an IgG-binding domainof streptococcal protein G, i.e. the C2 domain. In order toanalyze the contribution of the different amino acids to thealkaline sensitivity of the domain we used a single point mutationstrategy. Amino acids known to be susceptible towards high pH,asparagine and glutamine, were substituted for less-alkali-susceptibleresidues. In addition, aspartic acid residues were mutated toevaluate if the stability could be further increased. The stabilityof the different C2 variants was subsequently analyzed by exposingthem to NaOH. The obtained results reveal that the most sensitiveamino acid towards alkaline conditions in the structure of C2is Asn36. The double mutant, C2_N7,36A, was found to be the moststable mutant constructed. In addition to the increased alkalinestability and also very important for potential use as an affinityligand, this mutated variant also retains the secondary structure,as well as the affinity to the Fc fragment of IgG.