芽胞杆菌高效分泌表达异源蛋白的研究进展

外泌蛋白易于分离纯化,有利于以较低的生产成本获得大量的目标蛋白,因此分泌表达是一种理想的外源基因的表达方式。芽胞杆菌由于其具有较好的分泌能力,被认为是一种理想的表达外源基因的宿主。到目前为止已有大量来源于不同生物的外源基因在芽胞杆菌中实现了高效分泌表达。但是芽胞杆菌的分泌表达系统仍然存在很多问题,如对某些目的蛋白的分泌量低等,限制了其作为“细胞工厂”生产目标蛋白的应用。综述了芽胞杆菌的外泌系统,分析了芽胞杆菌分泌蛋白过程中的限制因素,并总结了相关的解决方案。     

苜蓿根瘤菌nodC蛋白在大肠杆菌中的过量生成和分离纯化

苜蓿根瘤菌（Ｒｈｉｚｏｂｉｕｍｍｅｌｉｌｏｔｉ）ｎｏｄＣ蛋白是结瘤基因ｎｏｄＣ编码的４３ｋＤ多肽（ＮｏｄＣ）。应用噬菌体Ｔ７ＲＮＡ聚合酶／启动子表达系统．ｐＴ７－５作为载体质粒．构建了带有ｎｏｄＣ基因的ＰＢＦ６克隆．经诱导在大肠杆菌ＪＡＫＥ中获得表达,过量生成ＮｏｄＣ,占细胞总蛋白量的５％。经细胞膜蛋白组份的分离,Ｂｉｏ－ｇｅｌ柱层析,ＳＤＳ－ＰＡＧＥ电泳等获得了比较纯化的ＮｏｄＣ。     

人纤溶酶原激活剂抑制物2型(PAI-2)在Pichia pastoris中的表达

用ＰＣＲ方法从ｐＰＡＩＪ．７中扩增人纤溶酶原激活剂抑制物２型（ＰＡＩ－２）基因,与ｐＰＵＣ１８重组,经限制性内切酶片段分析与核苷酸序列分析,获得全长人ＰＡＩ－２基因．ＰＡＩ－２基因与表达载体ｐＰＩＣ９重组,构建受乙醇氧化酶１基因（ＡＯＸ１）启动子与转录终止区控制的酵母表达质粒,转化ＧＳ１１５宿主菌,经表型筛选和ＰＣＲ扩增筛选阳性克隆,用甲醇诱导表达,重组ＰＡＩ－２以分泌型表达,占分泌总蛋白的３０％,具ＰＡＩ－２抗原性,与低分子量尿激酶形成了抗ＳＤＳ复合物,具抑制纤溶的活性（９１．４ＡＩＵ／ｍｌ）．对培养条件也进行了探讨．     

转基因烟草中Bt毒蛋白基因的表达行为

构建了高效植物表达载体ｐＢｉｎＭｏＢｃ,该载体携带超强表达复合启动子ＯＭ及Ω因子控制下的ＣｒｙＩＡ（ｃ）基因。采用根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导的方法转化烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）,ＥＬＩＳＡ检测表明,大多数转基因烟草中ＣｒｙＩＡ（ｃ）基因表达量均超过０．１％,最高可达０．２５５％;转基因烟草     

低温菌启动子分析及异源蛋白高效表达

在已构建的能在低温菌和E. coli中正常复制的启动子探针质粒的基础上, 筛选到了强启动子, 通过RT-PCR评估了启动子活性, 并通过引物延伸实验确定了转录起始位点和启动子核心序列。利用其中最强的启动子构建了低温蛋白表达质粒, 使一个热不稳定a-淀粉酶在低温下(7℃)得到了高效表达, 表达量达胞外总蛋白的35%。显示出该表达系统具有高效表达热不稳定蛋白质的潜在应用价值。     

低温菌启动子分析及异源蛋白高效表达

在已构建的能在低温菌和E. coli中正常复制的启动子探针质粒的基础上, 筛选到了强启动子, 通过RT-PCR评估了启动子活性, 并通过引物延伸实验确定了转录起始位点和启动子核心序列。利用其中最强的启动子构建了低温蛋白表达质粒, 使一个热不稳定a-淀粉酶在低温下(7℃)得到了高效表达, 表达量达胞外总蛋白的35%。显示出该表达系统具有高效表达热不稳定蛋白质的潜在应用价值。     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

霍乱毒素B亚基基因具有自己的启动子

本研究发现并证实霍乱毒素Ｂ亚基基因上游ＸｂａＩ～ＣｌａＩ限制性片段内存在具有启动子活性的序列;在该启动子作用下,霍乱毒素Ｂ亚基表达水平可达２００ｍｇ／Ｌ,氯霉素乙酰基转移酶基因表达水平随培养条件不同在０．３～１０ｍｇ／Ｌ之间,大肠杆菌β－半乳糖苷酶基因的表达量达４１００单位／ｍｌ。在该启动子的控制下霍乱毒素Ｂ亚基基因可以高效表达,该启动子的存在可能是霍乱毒素操纵子中霍乱毒素Ｂ亚基表达量是Ａ亚基的６倍的原因。     

新型大肠杆菌裂解系统在重组蛋白回收中的应用

溶菌酶通过降解细菌细胞壁的肽聚糖裂解细菌,利用此特性,构建温度敏感的内源裂解系统,达到高效释放和回收重组蛋白的目的。构建了温度敏感的Ｔ４溶菌酶基因表达质粒ｐＳＣ－ｌｙｓ（ｐＳＣ１０１复制子）和ｐ１５Ａ－ｌｙｓ（ｐ１５Ａ复制子）,质粒与工程菌构成了新型内源裂解系统,系统可以与高拷贝复制子（如ｐＭＢ１,ＣｏｌＥ１）的重组蛋白表达擀粒兼容。结果表明,裂解系统的量适裂解条件由三个要素构成：ｐＳＣ－ｌｙｓ－     

分泌载体pUS186的构建及地衣杆菌α－淀粉酶基因在枯草杆菌中的表达和分泌

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克隆了枯草杆菌染色体的启动子和信号肽序列,将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ３１酶切,Ｔ４ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ⅱ及ｐＵＳＡ１８６Ⅰ系列质粒,将这些重组质粒转化枯草杆菌ＱＢ１１３０（ａｍｙ－）后都能向胞外分泌淀粉酶,酶活测定结果表明,基因表达水平比用原有的启动子高１－２倍,蛋白质分泌率在８４－９６％之间。     

Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum.

By using appropriate Corynebacterium glutamicum-Escherichia coli shuttle plasmids, the gene encoding the fibronectin-binding protein 85A (85A) from Mycobacterium tuberculosis was expressed in C. glutamicum, also an actinomycete and nonsporulating gram-positive rod bacterium, which is widely used in industrial amino acid production. The 85A gene was weakly expressed in C. glutamicum under the control of the ptac promoter from E. coli, but it was produced efficiently under the control of the promoter of the cspB gene encoding PS2, one of the two major secreted proteins from C. glutamicum. The 85A protein was produced in various forms, with or without its own signal sequence and with or without the signal sequence and the NH2-terminal (18-amino-acid) mature sequence of PS2. Western blot analysis with monoclonal antibodies raised against the M. tuberculosis antigen 85 complex showed that recombinant 85A protein was present in the corynebacterial cell wall extract and also released in extracellular culture medium. NH2-terminal microsequencing of recombinant 85A secreted by C. glutamicum showed that signal peptide was effectively cleaved off at the predicted site. The recombinant 85A protein was biologically active in vitro, inducing significant secretion of Th1 T-cell cytokines, particularly interleukin-2 and gamma interferon, in spleen cell cultures from mice vaccinated with live Mycobacterium bovis BCG. Heterologous expression of mycobacterial antigens in C. glutamicum now offers a potent tool for further immunological characterization and large scale preparation of these recombinant proteins.     

Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells

An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.     

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli

Abstract Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (lon protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.     

Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure.

An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli. Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E. coli thioredoxins. The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously. Anomalous scattering factors were deduced from synchrotron X-ray absorption measurements of crystals of the selenomethionyl proteins. Taken with reference to experience in the structural analysis of selenobiotinyl streptavidin by the method of multiwavelength anomalous diffraction (MAD), these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of atomic structures.     

Cloning of Yersinia pseudotuberculosis calcium-dependence plasmid pYV6953 BamHI fragments and their analysis in Escherichia coli mini-cells

Calcium dependence plasmid pYV6953 (70.4 kb) in Yersinia pseudotuberculosis cells codes for the major quantities synthesis of 150; 48.5; 19.4 Kd outer membrane proteins and the 51, 38, 27 Kd proteins secreted into the culturing medium. These outer membrane and secreted proteins are synthesized in considerable amounts in Yersinia pseudotuberculosis strains 6953 and 9547 at 37 degrees C and in the absence of calcium ions in the culturing medium. BamHI fragments of the plasmid pYV6953 as components of the recombinant plasmids code for the synthesis of 150; 66.6; 51; 48.5; 47; 38 and 21.5 Kd proteins in Escherichia coli mini cells. The synthesis of 150 and 48.5 Kd proteins is determined by the BamHI fragment 9 of the plasmid pYV6953 (3.3 kb). Addition of up to 8% of ethanol inhibiting the protein synthesis eliminates the 150 Kd protein but not the 48.5 Kd synthesis. The 48.5 Kd protein is concluded to be a subunit of the 150 Kd protein. The plasmid pYV6953 is different from the known plasmids pIB1 and pCD1 plasmids as far as the outer membrane and secreted proteins coded by the plasmids are concerned.     

广西眼镜王蛇毒酸性磷脂酶A2-1在不同载体的表达

目的构建广西眼镜王蛇毒酸性磷脂酶A2-1(APLA2-1)在不同载体的重组表达质粒,在E.coli中表达APLA2-1并比较不同表达系统对APLA2-1的表达效果。方法将广西眼镜王蛇毒酸性磷脂酶A2-1(AP-LA2-1)基因克隆至表达载体pBLMVL2和pET28a( ),分别转化入大肠杆菌RR1和BL21,经过诱导表达,应用SDS-聚丙烯酰胺凝胶(SDS-PAGE)及Western blot观察重组蛋白表达情况。结果成功构建了重组质粒pBLMVL2-APLA2-1和pET28a-APLA2-1。pBLMVL2-APLA2-1在SDS-PAGE上没见明显表达带,在Western blot上可见一14 kD的表达带。pET28a-APLA2-1在SDS-PAGE上有一明显的18 kD表达条带,表达产物AP-LA2-1约占细菌总量30%,并以包涵体的形式存在。结论APLA2-1可在大肠杆菌中表达,pET28a( )对APLA2-1的表达效果优于pBLMVL2。     

Expression in Escherichia coli of hybrid genes containing sequences coding the bovine adrenocorticotropic hormone

E. coli strains producing a hybrid protein, containing adrenocorticotropic hormone (ACTH) and protein A of S. aureus was obtained. The sequence coding for ACTH was obtained from the bovine proopiomelanocortin cDNA and, after the modification of the 5'- and 3'-terminal parts, was linked with the protein A gene and its derivatives due to synthetic adaptors. Three forms of ACTH gene, coding this hormone with differing N-terminal amino acid were used to construct the fusion gene. The hybrid proteins contain Asp-Pro or (Asp)4-Lys sequences for obtaining ACTH by acid or enterokinase treatment, respectively. It is shown that each of the constructed plasmids direct the synthesis of hybrid protein in E. coli. This protein was purified by the use of IgG-sepharose. The level of the expression of the hybrid protein is 4 mg/l of the bacterial culture. Most of the synthesized protein is secreted into the periplasmic space.