人乙醛脱氢酶2的原核表达及条件的优化

通过双酶切、连接转化等方法将人乙醛脱氢酶2(ALDH2)基因克隆至载体pGEX-4T-1上,构建原核表达重组质粒pGEX-4T-1-ALDH2。重组质粒转化至E.coli BL21(DE3)后经IPTG诱导,SDS-PAGE分析表明目的蛋白得到大量表达。由于此载体本身带有融合蛋白标签GST(谷胱甘肽转移酶),所以得到了部分可溶性的目的蛋白。通过对表达条件进行探讨,发现在28℃下,以终浓度0.1mmol·L-1的IPTG诱导表达10h,可溶性的目的蛋白约占总蛋白的25%。表明,融合蛋白标签GST及较低的诱导温度有利于提高人乙醛脱氢酶2基因在大肠杆菌内的可溶性表达。     

人乳头瘤病毒16型L1蛋白VLP的原核表达及纯化

目的在大肠杆菌中表达人乳头瘤病毒16型(HPV16)L1蛋白病毒样颗粒(VLP),并进行纯化,为研制宫颈癌疫苗提供新的思路。方法以HPV16基因组DNA为模板,PCR扩增HPV16L1基因的编码区,定向克隆至原核表达载体pGEX-4T-1中谷胱甘肽转移酶(GST)编码区下游,构建重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot鉴定后,采用GST纯化柱纯化重组融合蛋白,并用凝血酶切去GST标签,再经S100分子筛纯化,浓缩后,电镜观察纯化蛋白的VLP形态。结果重组表达质粒经双酶切和测序,证明构建正确。表达产物的相对分子质量约为80000,主要以可溶性形式存在。Western blot显示,目的蛋白可与小鼠抗HPV16L1抗体发生特异性反应。纯化蛋白的纯度约为95%,在电镜下可见直径约为50～60nm的VLP。结论已成功地在大肠杆菌中表达了可溶性的HPV16L1蛋白VLP,并进行了纯化,为宫颈癌疫苗及血清学诊断试剂的研制奠定了基础。     

重组Echistatin融合基因工程菌高密度发酵工艺优化

目的优化大肠杆菌表达重组蛇毒锯鳞蝰素(Echistatin,Ecs)融合基因工程菌的发酵工艺。方法在15L发酵罐内,研究培养基、培养条件和诱导时间对工程菌生长和目的蛋白表达的影响,并考察工程菌中重组质粒的遗传稳定性。结果工程菌在pH7·4的2×YT培养基中诱导4h,菌体湿重可达75g/L,目的蛋白表达量约占总蛋白的35%,所构建的重组质粒在BL21宿主菌中传代稳定。结论优化了Ecs融合基因工程菌的发酵和表达条件,为规模化生产奠定了基础。     

胸腺素α1的基因合成、表达及活性测定

目的　合成胸腺素α1(Tα1)基因并在原核细胞中表达。方法　用半酶半化学合成并克隆Tα1基因 ,将该基因插入表达质粒pGEX 4T 1并在大肠杆菌进行表达 ,对表达的融合蛋白GST Tα1利用亲和层析法纯化 ,再经Thrombin酶切后 ,获得重组Tα1进行生物学活性检测。结果　经测序证实合成的Tα1序列与设计的一致。构建的重组表达载体pGEX 4T 1 Tα1在大肠杆菌中得到高效表达。E玫瑰花结形成试验证明重组Tα1具有生物学活性。结论　合成的Tα1基因在大肠杆菌中表达出具有生物活性的蛋白。     

基因工程菌株BLG8900的遗传稳定性

目的研究基因工程菌株BLG8900的遗传稳定性。方法在有选择压力(加氨苄西林)的条件下,测定pYC2质粒在大肠杆菌BLG8900株[含有质粒pYC2的BL21(DE3)]中的遗传稳定性。结果工程菌连续传10、25、50和100代后质粒具有良好的稳定性,而且经BamHⅠ/EcoRⅠ双酶切,酶切图谱相同。第100代菌株重组质粒的GnRH/TRS序列与原代菌株相同。原代与第10、25、50和100代菌株经IPTG诱导表达,GST-GnRH/TRS融合蛋白表达水平、菌体蛋白的SDS-PAGE图谱鉴定无明显差异。Western blot表明各代菌株表达产物均具有GnRH抗原特异性。结论质粒pYC2在大肠杆菌BL21(DE3)宿主菌中有较好的遗传稳定性。     

Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone

The alpha-subunits of human hemoglobin (Hb) have been more difficult to express than beta-chains owing to the high instability of alpha-chains. Here, we describe the production in Escherichia coli of a soluble recombinant alpha-Hb with human alpha-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-alpha-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express alpha-hemoglobin and human AHSP. While the GST-alpha-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-alpha-AHSP permits the co-expression of soluble GST-alpha-Hb and GST-AHSP. The alpha-Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant alpha-Hb complex were similar to those of recombinant AHSP/native alpha-Hb complex as assessed by UV/visible and CO or O(2) binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.     

猪圆环病毒2b亚型Cap融合蛋白的原核表达及其免疫原性分析

目的原核表达猪圆环病毒2b亚型(porcine circovirus subtype 2b,PCV2b)Cap融合蛋白,并分析其免疫原性。方法以PCV2毒株(CAU0673)基因组为参考序列,His-Sumo-PCV2b-Cap-pUC57重组质粒为模板,设计特异性引物,PCR扩增目的片段;将其产物克隆至pMAL-C4x载体中,构建pC4x-PCV2b-Cap重组质粒,转化至大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物进行直链淀粉树脂纯化。SDS-PAGE分析目的蛋白的可溶性,Western blot检测其反应原性;以纯化的融合蛋白免疫BALB/c小鼠,Western blot检测小鼠血清抗体特异性。结果经双酶切及测序鉴定,重组质粒pC4x-PCV2b-Cap构建正确;融合蛋白MBP-Cap(matlose binding protein Cap)最适诱导条件为1 mmol/L IPTG 37℃诱导6 h,其以可溶性形式存在,相对分子质量约为81000,与PCV2b阳性猪血清、兔抗MBPtag多克隆抗体及免疫小鼠抗血清均可发生特异性反应。结论成功构建了pC4x-PCV2b-Cap重组表达载体,获得了可溶性的MBP-Cap蛋白,其纯化蛋白具有较好的免疫原性。本文为PCV2b诊断试剂盒的研发奠定了理论基础。     

人IL-23R胞内区基因的克隆与原核表达

目的克隆人IL-23R(hIL-23R)胞内区基因,并在大肠杆菌中表达融合蛋白。方法通过PCR获得hIL-23R基因的胞内区片段,克隆至载体pGEX-4T-1中,构建重组原核表达质粒pGEX-4T-1-hIL-23R(I),转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE及Westernblot进行鉴定。结果hIL-23R胞内区基因的PCR扩增产物经琼脂糖凝胶电泳分析可见约760bp的目的片段。重组原核表达质粒经双酶切及测序证明构建正确。表达的融合蛋白相对分子质量约为55000,在低温(20℃)、低IPTG浓度(0.5mmol/L)诱导条件下能以可溶形式表达,可溶性蛋白的表达量约占菌体可溶性蛋白总量的16.1%,且可被兔抗GST多克隆抗体识别。结论已成功克隆了hIL-23R胞内区基因,并在大肠杆菌中表达了可溶性的GST融合蛋白。     

Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment

We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library.     

Expression and enzymatic characterization of the soluble recombinant plasmepsin I from Plasmodium falciparum

The plasmepsins are involved in the degradation of host cell hemoglobin during malaria infection. Plasmepsin I (PM I) initiates the degradative process, and has been suggested as an attractive target for the treatment of malaria. The production of active recombinant PM I, however, has been challenging. We report for the first time, the expression and partial characterization of soluble recombinant PM I from Plasmodium falciparum in which a truncated form of PM I (Lys77P-Leu329) (P indicates a propart residues) was fused to thioredoxin in the pET32b(+) vector, Trx-tPM I and expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The soluble fusion protein was purified from cell culture using a combination of Ni(2+) affinity and gel filtration chromatography and was capable of autocatalytic activation at pH 4.0-5.5, which occurred at Leu116P-Ser117P, seven residues upstream of the native cleavage site (Gly123P-Asn1). The mature tPM I (mtPM I) was capable of hydrolyzing both human hemoglobin with a pH optimum of pH 2.8-4.0 and the synthetic fluorogenic peptide EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH with a dual pH optima of pH 2.5-3.0 and pH 4.5-5.5. Using the synthetic substrate, mtPM I exhibited kinetic parameters comparable to native PM I.     

人溶菌酶-抗菌肽tachyplesins融合蛋白的原核表达及其抗菌活性

目的原核表达人溶菌酶(Human lysozyme,hLYZ)和抗菌肽tachyplesins融合蛋白,并检测其抗菌活性。方法人工合成抗菌肽tachyplesins基因和linker,与pMD18-T-hLYZ上切下的hLYZ基因融合,将融合基因克隆至带有GST标签的原核表达载体pGEX-4T-1上,转化大肠杆菌BL21(DE3),IPTG诱导表达,并对表达条件进行优化。表达的融合蛋白经亲和层析纯化后,进行抗菌活性检测。结果重组表达质粒pGEX-4T-1-hLYZ-tachyplesins经PCR、双酶切及测序鉴定,证明构建正确;表达产物经SDS-PAGE分析,在相对分子质量约44000处可见目的蛋白条带,诱导温度在25℃,IPTG终浓度为0.8mmol/L,诱导时间为6h,融合蛋白表达效果较好,主要以可溶性形式存在;纯化的融合蛋白纯度可达90%以上,对金黄葡萄球菌和大肠杆菌具有一定的抑制作用。结论已成功在原核细胞中表达了人溶菌酶-抗菌肽tachyplesins融合蛋白,纯化的融合蛋白具有一定的抗菌活性。     

人甲状旁腺素N-端34肽基因的合成、克隆和融合表达

目的构建重组人甲状旁腺素N-端34(PTH34)肽的大肠杆菌融合表达菌株,并对其表达产物进行检测。方法选用大肠杆菌偏爱密码子,设计优化的人甲状旁腺素N-端34肽基因序列,通过6个DNA片段分段合成、片段连接、PCR扩增,经TA克隆到pMD18载体中,经测序验证,构建GST融合表达载体。结果经SDS-PAGE检测PTH(1-34)肽与GST蛋白的融合表达,经Westernblot分析具有免疫活性,通过GST亲和层析和Xa因子酶切,利用小分子蛋白电泳可检测到PTH(1-34)肽。结论已获得了能表达GST-PTH(1-34)融合蛋白的菌株。表达产物具有免疫活性,且能被Xa因子消化得到PTH(1-34)肽。     

人溶菌酶基因的原核表达及其生物学活性

目的在大肠杆菌中表达人溶菌酶(hLYZ)基因,并检测其生物学活性。方法将人溶菌酶基因克隆至带有GST融合标签的原核表达载体pGEX-4T-1上,转化大肠杆菌BL21(DE3),经IPTG诱导表达。表达产物经亲和层析纯化后,进行Western blot鉴定,并用溶壁微球菌比浊法检测酶活性,热激法检测其生物学活性。结果经PCR、双酶切鉴定和核苷酸序列测定,表明重组表达质粒pGEX-4T-hLYZ构建正确。SDS-PAGE显示,在相对分子质量约40000处可见目的蛋白条带,表达量占菌体总蛋白的46.5%,目的蛋白在裂解沉淀中占17.0%,在裂解上清中占58.3%,主要以可溶形式表达。纯化后,重组蛋白纯度可达95%以上,且具有良好的反应原性。重组hLYZ酶活性为2389U/mg,对金黄葡萄球菌和大肠杆菌具有抑制作用。结论已成功地在大肠杆菌中表达了可溶性重组人溶菌酶,且具有较高的生物学活性。     

蜂毒肽/变构hIL-2融合蛋白的原核表达及其对宿主菌生长的影响

目的构建蜂毒肽(Melittin)与变构hIL-2融合基因原核表达质粒,并检测表达的融合蛋白对宿主菌E.coli生长的影响。方法以质粒pGEX-4T-2/Melittin-IL-2(88Arg)为模板,通过PCR定点诱变为Melittin-IL-2(88Arg,125Ala),将PCR产物和pET-15b载体分别经双酶切后连接,构建重组表达质粒pET-15b/M-IL-2(88Arg,125Ala),转化E.coliBL21(DE3),IPTG诱导表达。SDS-PAGE及ELISA分析表达产物;检测诱导不同时间重组菌的A600值,绘制生长曲线,并进行活菌计数。结果PCR扩增的目的片段长542bp,重组表达质粒测序分析证明目的基因如预期突变;ELISA可检测到目的蛋白表达,但表达量较低;SDS-PAGE分析未见目的条带;诱导表达4h,重组菌A600值由0.8下降至0.6;诱导2h,活菌计数由108个/ml降至104个/ml。结论已成功构建了原核表达质粒pET-15b/M-IL-2(88Arg,125Ala),表达的融合蛋白可能对宿主菌具有毒性,从而杀伤宿主菌。     

A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli

Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.     

金黄葡萄球菌野生株肠毒素B重组蛋白的表达及纯化

目的在大肠埃希菌中表达金黄葡萄球菌野生株肠毒素B(SEB)重组蛋白,并进行纯化。方法采用PCR方法从金黄葡萄球菌基因组中扩增出SEB基因片段,与pMD18-T载体连接,构建重组质粒pMD18-T/SEB,测序分析后,亚克隆入表达载体pGEX-4T-2,转化感受态E.coliJM109,IPTG诱导表达。表达产物采用B-PER GST Fusion Protein Purification Kit纯化后进行Western blot鉴定。结果SEB基因扩增片段大小为705bp,测序结果显示与金黄葡萄球菌S6株序列相比无碱基的插入或缺失,同源性为98.4%,存在11个碱基变异,其中7个为无义突变,4个为有义突变(突变位点位于第364、699、899和988位,编码氨基酸变化分别为:Ser→Ala、Gln→His、Asn→Ser和Met→Leu);表达的含GST的融合蛋白相对分子质量约53000,纯化后经SDS-PAGE分析,可见单一条带。Western blot显示表达产物可被兔抗SEB抗体所识别。结论已成功表达并纯化了金黄葡萄球菌野生株SEB重组蛋白,为开发SEB免疫诊断试剂提供了材料。     

重组质粒DNA D-GPEi工程菌发酵工艺的优化

目的优化重组质粒D-GPEi工程菌发酵工艺条件。方法控制相关发酵参数,观察溶解氧浓度、比生长速率和培养时间对工程菌生长和质粒浓度的影响。结果发酵培养对数生长期溶解氧浓度控制在30%~50%。补料前比生长速率为0.5~0.7/h,6 h开始补料,比生长速率下降,12 h后比生长速率为0.1~0.2/h。连续发酵3批工程菌,21 h后收集菌体,得到菌体平均产量为89.4 g/L(A=52.6),质粒平均产量为359.8 mg/L。3批工程菌浓度和质粒拷贝数差异无显著意义。结论此发酵工艺得到工程菌浓度和质粒拷贝数较高,重复性好,适用于大规模工业化生产。     

人心肌肌酸激酶MB同工酶的原核表达及纯化

目的原核表达并纯化人心肌肌酸激酶MB同工酶(creatine kinase MB,CK-MB)。方法分别从含CK-M、CK-B基因片段的质粒SC319293和SC119007中扩增CK-M、CK-B基因,构建重组原核表达质粒pGEX-4T-1-CK-MB,转化大肠埃希菌BL21(DE3),IPTG诱导表达重组蛋白,经GST亲和层析柱纯化后,ELISA法检测其抗原性。结果重组表达质粒pGEX-4T-1-CK-MB经菌落PCR、双酶切及测序鉴定,证明构建正确;表达的重组CK-MB蛋白相对分子质量约110 000,表达量占菌体总蛋白的57%,主要以包涵体形式存在;纯化的CK-MB蛋白纯度大于90%,可与兔抗CK-B、CK-M多克隆抗体特异性结合。结论成功原核表达并纯化了重组CK-MB蛋白,为其大量生产奠定了基础。