运用均匀设计法优化rPA(K)在原核系统中的表达条件

运用均匀设计法对影响rPA(K)在大肠杆菌中表达的因素进行了优化设计,摸索出了对表达产物进行诱导表达的最佳优化表达条件。结果如下：溶解氧为90%,IPTG诱导时间为5.3 h、IPTG终浓度为0.1 mmol/L、培养基为HD、pH值为6.0、诱导温度为25?C。目的蛋白平均密度从0.16提高到0.48,是优化前的3倍。     

重组人纤溶酶原Kringle1-5的制备及其

为了研究重组人纤溶酶原 Kringle1-5(K1-5)的抗血管生成活性及其对内皮细胞增殖的影响, 通过PCR扩增人纤溶酶原K1-5 cDNA,定向克隆于原核表达载体pET30a(+)中,构建重组表达载体pET-K1-5, 转化E.coli BL21(DE3), IPTG诱导表达,SDS-PAGE 和Western 杂交检测K1-5的表达。鸡胚尿囊膜 (CAM) 实验和MTT实验分别检测重组人纤溶酶原Kringle1-5对鸡胚新生血管生成和内皮细胞的抑制作用。结果表明,IPTG诱导原核表达载体pET-K1-5在E.coli BL21(DE3)中的表达量约占菌体总蛋白量的32%, K1-5主要以包涵体形式存在,包涵体经过洗涤、溶解、Ni-spin 亲合柱层析纯化以及蛋白质复性等步骤后,获得了纯度约为96%的重组K1-5蛋白。CAM实验表明,原核表达的重组人K1-5能有效地按剂量依赖的方式抑制鸡胚新生血管的形成。MTT实验结果显示,重组人K1-5特异地抑制内皮细胞的增殖, 而对非内皮细胞无抑制作用。     

人呼吸道合胞病毒截短F1重组蛋白包涵体的制备、纯化和复性

目的将前期在大肠埃希杆菌中获得表达的A型人呼吸道合胞病毒兰州分离株截短的F1重组蛋白进行纯化和复性,为后期动物免疫制备抗原。方法 37℃诱导重组菌体p ET-42b-F1J/Rossata,诱导完毕后离心收集菌体,高压破碎菌体并收集包涵体后用不同浓度的Triton X-100(细胞裂解液)洗涤包涵体3次。洗涤的包涵体用8 mol/L尿素进行溶解并用镍离子亲和层析方法进行初步纯化,用阳离子交换层析方法对初步纯化蛋白进行最终的纯化。亲和层析纯化蛋白用3种不同的复性液进行了稀释复性。结果 37℃诱导5 000 m L重组菌p ET-42b-F1J/Rossata共收获37 g湿菌体,经过不同浓度Triton X-100洗涤包涵体后纯度可达75%。包涵体用8 mol/L尿素溶解后经镍离子亲和层析纯化纯度约为40%,再用阳离子交换层析介质SP HP进一步纯化样品后纯度可达90%。纯化蛋白以3种不同的复性液都能得到复性,其中复性液3的复性效果相对较好。结论实验中探索了人呼吸道合胞病毒截短F1重组蛋白包涵体的纯化方法及步骤,为后期的蛋白制备及动物免疫奠定了基础。     

重组人表皮生长因子包涵体的表达、纯化及复性

目的:通过优化人表皮生长因子(hEGF)基因序列,利用大肠杆菌大量表达重组hEGF(rhEGF)包涵体,经过包涵体纯化复性获得高活性的rhEGF。方法:采用全基因合成优化后的序列,克隆至pET-30a表达载体中,转化大肠杆菌BL21(DE3),经IPTG诱导表达,将rhEGF包涵体用尿素溶解后过Ni柱纯化并稀释复性,根据药典对得到的rhEGF进行纯度及活性测定。结果:构建了rhEGF的表达载体pET-30a-rhEGF,表达出的蛋白主要存在于包涵体中,相对分子质量为6.5×10~3,包涵体经过纯化复性后获得的rhEGF纯度可达92.8%,生物活性约4.94×10~7IU/mg。结论:得到了具有较高活性的rhEGF。     

HER2胞外段的克隆与原核表达

应用RT-PCR技术从人乳腺癌细胞系SK-BR-3中克隆出人表皮生长因子受体2(human epidermal growth factorreceptor 2,HER2)基因的胞外段,并插入到表达载体pET-30a中,得到重组表达载体pET30-HER2(Ex)。将该载体转化至大肠杆菌BL21(DE3)细胞中,加入IPTG进行诱导表达,成功获得HER2胞外段蛋白。分别提取培养液上清、大肠杆菌周质腔、细胞质可溶性及不可溶性组分蛋白进行SDS-PAGE电泳分析,确定目的蛋白定位于大肠杆菌细胞质包涵体中。通过改变诱导温度、诱导物浓度、诱导起始菌体密度和诱导时间,寻找最佳表达条件,使目的蛋白的表达量达到最高。结果表明,在37℃下,OD600达到1.0时,经终浓度为0.1 mmol/L的IPTG诱导4 h,目的蛋白的表达量最高。将重组表达菌进行超声破碎,分离出包涵体组分,经Ni2+亲和层析纯化后获得了纯度>90%的HER2胞外段蛋白,从而为抗HER2抗体的制备及肿瘤疫苗的研究奠定了基础。     

单纯疱疹病毒1型囊膜糖蛋白gD在大肠杆菌中的表达、纯化及其免疫活性的鉴定

目的:在大肠杆菌中表达1型单纯疱疹病毒(HSV-1)囊膜糖蛋白gD,纯化重组蛋白并对其免疫活性进行鉴定。方法:将HSV-1 gD 基因克隆入原核表达载体pET-28b,利用异丙基-B-D-硫代吡喃半乳糖苷(IPTG)诱导重组质粒转化的大肠杆菌,探讨IPTG浓度、诱导时间、诱导温度对重组蛋白表达的影响;盐酸胍裂解变性包涵体,镍柱亲和层析法纯化gD蛋白,并对纯化后的蛋白进行透析复性;Western blot和ELISA检测gD蛋白的免疫活性。结果:酶切和测序结果表明gD基因克隆入pET-28b载体。该重组质粒转化的大肠杆菌经IPTG诱导后重组蛋白主要以包涵体形式存在,大小约40kDa。gD蛋白诱导表达的最佳条件为0.5mmol/L IPTG于37℃诱导8h。镍柱亲和层析法纯化获得的gD蛋白总量为3.1mg/L,透析复性后获得的gD蛋白总量为1.3mg/L,复性率为41.37%。Western blot及ELISA检测表明表达的gD蛋白具有免疫活性。结论:在大肠杆菌中表达并纯化获得具有免疫活性的HSV-1 gD蛋白,为进一步制备HSV-1诊断试剂和预防疫苗奠定了基础。     

苦荞过敏蛋白TB22的原核表达及纯化

为了确定苦荞主要过敏原蛋白TB22的抗原决定簇,揭示其致敏机制,为以后的分子改造及育种改良打下基础,需要在体外微生物体系中获得大量的纯化蛋白。以pQE31为表达载体,M15为表达菌株,使用IPTG诱导,在37℃获得了以包涵体形式存在的表达产物。经WesternBlotting检测,证明表达条带N端带有Histidinetag。使用8molL尿素初步纯化后,目的蛋白含量达到40%以上。进一步HitrapChelatingHP亲和纯化,表达蛋白的纯度达到90%以上。建立了适合重组TB22纯化的基本方法,得到了纯化的包涵体,为抗TB22抗体的制备及其抗原决定簇的研究奠定了基础。     

MMP9-PEX的原核表达、纯化与复性研究

目的 对人基质蛋白酶9（matrix metalloproteinase,MMP9）C端血红素结合蛋白样结构域（hemopexin domain,PEX）的原核表达条件、透析复性条件进行优化,以获得高表达的活性蛋白.方法 将构建好的原核表达载体PET-his-MMP9-PEX转化至大肠埃希菌菌株BL21（DE-3）,异丙基β-D硫代半乳糖苷（IPTG）诱导后产生包涵体蛋白,探讨不同IPTG浓度、不同诱导时间、不同菌液密度值对目的 蛋白表达产量的影响;盐酸胍裂解包涵体,采用NI-NTA进行纯化,对纯化蛋白的透析复性条件进行优化;采用明胶酶谱的方法检测MMP9-PEX的活性.结果 经纯化透析复性后蛋白的纯度在95%以上,复性回收率为36.86%.结论 本研究优化了MMP9-PEX的原核表达、透析复性条件,获得了有活性的MMP9-PEX蛋白,为进一步研究MMP9-PEX的功能提供了实验基础.     

大肠杆菌表达Trx-rPA融合蛋白的复性纯化

大肠杆菌高密度发酵以包涵体形式表达融合蛋白Trx-rPA,表达量22%。包涵体蛋白洗涤后经金属螯合层析纯化,纯度达80%以上。经胱氨酸衍生,以脉冲加样形式复性,复性率可高达30%。经ETI-Sepharose纯化,复性的融合蛋白生物活性可达3.5×105IU/mgPr.。融合蛋白可被rEK酶切释放rPA,酶切效率达85%以上。酶切液经IDA-Sepharose和SP-Sepharose层析纯化,rPA纯度达98%以上,生物活性50万IU/mgPr.。1L发酵液经分离、复性及纯化后,可得高纯度rPA300mg以上。     

次级淋巴组织趋化因子（SLC）基因克隆及在大肠杆菌中的表达

次级淋巴组织趋化因子(SLC)是通过搜索表达序列标签(EST)数据库克隆出来的一CC类趋化因子。以人SLC序列为蓝本,利用重叠PCR(SOE-PCR)的方法获得了适宜在大肠杆菌中表达的SLC基因,将此序列分别克隆至表达载体pTMF和pALM中,转化大肠杆菌,诱导表达。Western Blotting鉴定结果表明目的蛋白以可溶蛋白和包涵体两种形式表达,两种形式的蛋白所占比例依培养和诱导条件的不同而变化。对两种形式的表达产物分别用Ni-NTA金属亲和层析和包涵体复性方法纯化在实验中还对纯化条件进行了探索。对纯化蛋白的电泳结果显示:纯化样品的分子量比预期的分子量要大。     

融合牛肠激酶轻链EKL包涵体蛋白的复性纯化

大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEKL,主要以包涵体形式存在。包涵体经4mol/L尿素和0.5%TritonX-100洗涤,以6mol/L盐酸胍、100mmol/LDTT溶解,在胱氨酸存在下,以脉冲加样方式复性。融合蛋白复性在6mmol/L胱氨酸存在下、脉冲加量0.03mg/mL和复性终蛋白浓度0.3mg/mL为最佳复性方案。复性的融合蛋白加2mmol/LCaCL2后快速自切。经IDA-Sepharose及Q-Sepharose纯化,rEKL纯度可达95%以上,可高效酶切重组瑞特普酶融合蛋白Trx-rPA。实现了大规模生产rEKL,每升发酵液经复性及纯化后,可得rEKL60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。     

Refolding of the Fusion Protein of Recombinant Enterokinase Light Chain rEKL

The fusion protein of enterokinase light chain, DsbA-rEK_L, was expressed mainly in the inclusion body in E. coli. The recombinant bacteria were fermented to high density, with high expression of the fusion protein. After being washed with 0.5 % Triton X-100 and 4 mol/L urea, the inclusion body was dissolved in 6 mol/L guanidine and 100 mmol/L DTT, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03 mg/mL of fusion protein until its final concentration reached 0.3 mg/mL. The refolded protein was autocleaved, and the active EK_L molecule was released after the addition of 2 mmol/L of CaCl₂. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEK_L was found to be above 95 %, with a high activity to cleave the recombinant reteplase fusion protein, Trx-rPA. The yield of purified rEK_L was more than 60 mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large scale in a way such as expressed in the form of fusion proteins.     

Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol

The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and β-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20mM β-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90mg/l of the culture or 11.8 and 9.37mg/g of wet weight of cells in pQE30UA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE30UA as compared with purification by conventional methods.     

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Refolding and Purification of Zymomonas mobilis Levansucrase Produced as Inclusion Bodies in Fed-Batch Culture of Recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE–Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Improving purification of recombinant human interferon gamma expressed in Escherichia coli; effect of removal of impurity on the process yield

Process development and optimization studies were performed in order to improve the purification process of (rhIFN-gamma). The objective was to generate material with higher purity and quantity. An in-process control screening was developed to obtain the optimal condition for column chromatographic purification by measuring LPS, nucleic acids, rhIFN- gamma, monomer and its covalent dimers. A new resin screening method was applied to select optimal resin for each of the chromatographic columns. The resulting process used Butyl and Q-Sepharose, refolding and SP-Sepharose for purification of IFN-gamma. Effects of different process conditions such as cell lysis, removal of impurity and oxygen concentration were evaluated. Removal of impurities was evaluated by washing of inclusion bodies with 1% Triton X-100 and 3M urea and different chromatography steps. The results reveal that Triton removed about 43% of the LPS but urea had no effect on removal of nucleic acids and LPS. Further analysis show that removal of impurities by column chromatography decreases aggregation and increases the process yield. Oxygen concentration was identified as parameter that could have a significant impact on covalent dimers formation, as an unacceptable pharmaceutical form of rhIFN-gamma. On the basis of small-scale studies, optimum operating conditions were chosen and the purification process was successfully scaled-up to a pilot scale process with step yield and product quality that were better than previous reports.     

Optimization of phenol extraction procedures for preparation of RNA from mammalian lymphoid organs

We have investigated the effect of different concentrations of Triton X-100 on the resolution of microgram amounts of different hemoglobin subunit polypeptides during electrophoresis in polyacrylamide gels containing acetic acid, urea, and Triton X-100. The results of these studies indicate that adequate concentrations of Triton X-100 facilitate the resolution of polymorphic globin chains and that this type of electrophoretic separation is technically much simpler and more sensitive than currently used methods. Using this method, known and previously undescribed types of α and β chains can be detected. Furthermore, polyacrylamide gel slabs containing a horizontal gradient of Triton X-100 permit the identification of different globin chains present in lysates of erythrocytes or erythrocyte precursors without prior purification of the hemoglobins.     

Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca. 5U/mg was obtained.     

Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl₂). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.