大腹园蛛次壶腹腺丝的表达

基于大腹园蛛次壶腹腺丝Minor Ampullate Spidroin全长编码基因最新报道,研究了该基因的表达。利用PCR扩增该基因重复区一段长1 348 bp的片段P1,融合his-tag标签,构建酵母表达载体,在毕赤酵母菌GS115进行表达。同时构建大肠杆菌表达载体,在大肠杆菌BL21(DE3)中进行表达。SDS-PAGE和Western blotting检测结果表明,P1在两种表达系统中均可实现表达。研究结果显示:P1在GS115中的表达经优化后产量、产率有较大提高,且远高于BL21(DE3)中的表达,相应的纯化效率GS115也远高于对照BL21(DE3)的表达。研究表明酵母表达系统更适合重复度高、且富含Gly/Ala的天然蛛丝蛋白基因的表达,为表达全长天然MiSp编码序列提供前期实验基础,也为大规模蛛丝蛋白的重组表达建立了平台。     

Cloning and chromosomal mapping of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate-dehydrogenase from Arabidopsis thaliana

Both cDNA and genomic clones for the nuclear genes encoding chloroplast (cp) (gapA and gapB) and cytosolic (gapC) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana have been isolated and characterized. Genomic Southern-blot analyses indicate that there is only one copy of each gapA, gapB and gapC gene in A. thaliana. Comparison of the deduced amino acid (aa) sequences shows that the A and B subunits are highly similar (80% positional aa identity), while there is less similarity between the cp and cytosolic subunits (45% aa identity). These relationships are consistent with the idea that the cp and cytosolic GAPDHs evolved from different lineages, as suggested in our previous study of tobacco GAPDHs [Shih et al., Cell 47 (1986) 73-80]. In addition, the chromosomal locations for the three gap genes were determined by restriction fragment length polymorphism mapping; the three gap genes are not closely linked, gapA (55.8 cM) and gapC (0.0 cM) are on chromosome 3, and gapB (51.3 cM) is on chromosome 1.     

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.     

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 microg of extracellular alkaline, Ca(2+)-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The CDase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. CDase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular CDase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated CDase were expressed. High levels of intracellular CDase were expressed by Escherichia coli DH5alpha and E. coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E. coli DH5alpha culture, 280 microg of pure CDase was obtained after a three-step purification protocol. Under control of the T7-promotor CDase, without its signal peptide, was produced in inclusion bodies in E. coli BL21 cells. After refolding, 1.8 mg of pure active CDase was obtained from a 2.4-L culture after ammonium sulfate precipitation and gel filtration. Both the recombinant and wild-type CDases have a pH optimum of 8.5. The recombinant enzyme was partially characterized. This is the first report of a high yield CDase production system allowing detailed characterization of the enzyme at the molecular level.     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

Streptococcus agalactiae GAPDH is a virulence-associated immunomodulatory protein

Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

大鼠CRIP2蛋白的表达和分离纯化

目的:获得大鼠crip2基因片段,并在大肠杆菌中表达、纯化大鼠CRIP2(cysteine-rich intestinal protein 2)蛋白。方法:从大鼠主动脉组织中提取总DNA,RT-PCR扩增出相应大小的crip2 DNA片段,与pGEM-T-easy载体连接后测序;将测序正确的crip2按照BamHⅠ和HindⅢ酶切位点克隆入原核表达载体pRSET A,将连接产物转化大肠杆菌BL21,挑出阳性克隆,IPTG诱导表达重组的6×His融合蛋白,通过镍柱进行纯化。结果:PCR获得的crip2序列与GenBank报道的一致(为707 bp);重组融合蛋白在大肠杆菌BL21中以可溶形式高效表达,经SDS-PAGE和Western印迹分析,在相对分子质量为27×103处有特异的蛋白条带,经镍柱纯化后,得到了高纯度的CRIP2融合蛋白。结论:克隆了大鼠crip2基因片段,并在大肠杆菌BL21中高效表达,亲和层析纯化后获得高纯度的CRIP2融合蛋白。     

人睫状神经营养因子在大肠杆菌和毕赤酵母中的表达和生物活性测定

The recombinant human ciliary neurotrophi factor(hCNTF)expressed in E.coli aggregatedas inclusion bodies and refolding procedure was necessary to obtine the active protein.To overcome the disadvantage,we cloned hcntf gene into yeast expression plasmid pPIC9K and collected the plasmid pPIC9K-hcntf.Plasmid pPIC9K-hcntf was transformed into yeast Pichia pastoris GS115,and screened on G418-SD plates.The transformants with high copies of hcntf gene were inoculated into BMMY media and induced with 0.5% methanol.The recombinant hCNTF was secreted into the media.The amount of hCNTF in the supernatant was about 10 mg/L when incubated in the conical flasks and reached up to 60 mg/L under fed-batch condition in 15 L fermentator.The recombinant hCNTF expressed in E.coli was renatured as the control.The neonatal rat dorsal root ganglion assay showed that proteins expressed in both systems have the activity of promoting the growth of neuron axons.The phenomenon can be observed with only 3 μg hCNTF expressed in yeast present,which indicates that hCNTF was successfully expressed in Pichia pastoris and has a relatively high activity.     

大鼠热休克蛋白70的融合表达及纯化鉴定

目的：在大肠杆菌中高效表达并纯化大鼠热休克蛋白（HSP）70与麦芽糖结合蛋白（MBP）的融合蛋白,以进一步研究细胞外HSfr70的生物学功能。方法：用RT-PCR方法扩增目的基因,并将其克隆到原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序;将该重组表达载体转化大肠杆菌B121,用IPTG在不同温度及时间下进行诱导表达,建立最佳诱导表达条件;采用Amylose树脂预装柱对目的蛋白进行亲和纯化,并对不同表达条件下的产物进行SDS-PAGE及Westernblot分析。结果：克隆出目的基因,构建了融合表达载体pMAL-c2X／hsp70;诱导表达后经SDS-PAGE检测表明获得了目的条带,并纯化出纯度较高的融合蛋白;免疫印迹鉴定表明其具有抗原活性。结论：在大肠杆菌中高效表达并纯化了融合蛋白MBP-HSP70,为进一步研究细胞外HSP70的生物学效应提供了有用的材料。     

赤翅甲抗冻蛋白基因的原核表达及蛋白生物活性检测

根据GenBank中序列人工合成赤翅甲Dendroides canadensis的抗冻蛋白基因（afp）,将其克隆到载体pGEX-4T-1上,构建融合表达的重组质粒,转化大肠杆菌 BL21并进行原核表达。通过优化表达的诱导条件和SDS-PAGE检测,证明人工合成的赤翅甲抗冻蛋白基因能够特异性地表达,并以可溶性融合蛋白形式存在,相对分子质量约为40 kD。抗冻蛋白的生物活性检测表明,赤翅甲的抗冻融合蛋白能够提高细菌的耐寒能力。     

GAPDH基因克隆及其作为毕赤酵母内标物的可靠性研究

为了测定GAPDH在巴斯德毕赤酵母中作为内参蛋白的有效性,利用PCR鉴定巴斯德毕赤酵母中GAPDH基因,分别在含葡萄糖、甲醇、甘油培养基及不同温度下培养巴斯德毕赤酵母,采用SDS-PAGE、Western blotting和催化活性检测。测序结果与已报道的巴斯德毕赤酵母GAPDH基因完全一致。SDS-PAGE结果显示,表达蛋白分子量为35.4 kD,与预期分子量相符。Western blotting和催化活性测定显示,在葡萄糖诱导下GAPDH表达最高,甲醇次之,甘油最低。37℃下GAPDH表达略高于28℃。在同一诱导物或温度下,GAPDH表达不会随浓度或培养时间发生明显变化。结果表明,在同一种诱导方式下,GAPDH可以作为异源蛋白表达的内参对照。     

Characterization of group B streptococcal glyceraldehyde-3-phosphate dehydrogenase: surface localization,enzymatic activity,and protein-protein interactions

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent M(r) of approximately 173,500 migrating on a SDS--polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.     

基因工程手段提高磷脂酰丝氨酸合成酶活性的研究

为了提高目的蛋白磷脂酰丝氨酸合成酶(PSS)的表达,使用rTaq酶从E coli K12总DNA中PCR扩增获得磷脂酰丝氨酸合成酶基因片段,将其重组于高效表达载体pET28b质粒,转入相应表达菌株进行表达,收集菌体超声破碎获得含PSS的粗酶液,使用两相反应体系进行生物转化,HPLC-ELSD手段进行酶活检测。结果显示,重组菌株中PSS的酶活力比对照有所提高,同时获得酶活力提高的突变基因pssE210G。将突变基因重组于更高效的表达载体pBAD-MCS,转入相应宿主菌株表达。结果显示E.coli TOP10(pBAD-MCS-pss)表达的酶活性明显优于E.coli BL21(pET28b-pss)中表达的酶活性。以上实验结果表明,将目的基因重组于高效表达质粒有助于提高酶活力;组合到不同的表达质粒,酶活提高程度不同;磷脂酰丝氨酸合成酶基因第73位氨基酸发生突变,对其酶结构和酶活力有直接的影响。     

人角质化细胞生长因子-2基因的克隆、表达及产物的纯化、鉴定

用高表达菌株BL21codon plus compentent cells表达重组人角质化细胞生长因子(Hkgf-2)蛋白并初步纯化和检测其活性。通过RTPCR从流产胎儿肺组织中钓取hKGF-2cDNA,将其克隆入pBV220载体质粒。在大肠杆菌BL-21codon plus compent cells中表达hKGF-2蛋白。采用亲和层析和离子交换层析分离纯化,以细胞增殖实验测定表达蛋白的生物活性。结果显示,hKGF-2蛋白在BL21中得到高效表达;hKGF-2蛋白能刺激NIH3T3细胞的增殖,具有显著的促有丝分裂活性。     

Comparison of expression of monomeric and multimeric adenoregulin genes in Escherichia coli and Pichia pastorias

Adenoregulin is a 33 amino acid antibiotic peptide who belongs to dermaseptin family which is the first vertebrate family to show lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. Synthetic adenoregulin gene was cloned in 2, 4 and 6 tandem repeats and subcloned in pET32a and pET22b vectors. Recombinant plasmids were transformed into E. coli BL21(DE3), Fusion proteins of Trx-ADR1, Trx-ADR2 and Trx-ADR4 could be expressed after the hosts were induced by IPTG, but the expression level decreased dramatically with the number of tandem repeats increased. ADR1, ADR4 and ADR6 could not be expressed by E. coli without carrier proteins. But for Pichia pastoris GS115, ADR1 and ADR6 in the fermentation broth of the hosts could be detected by ELISA, and the bactericidal activities could also be observed.     

诺卡氏菌腈水合酶基因在重组毕赤酵母中的表达

为从基因水平上改造腈水合酶,进行了诺卡氏菌腈水合酶基因的外源表达研究。在重组大肠杆菌表达系统内,腈水合酶的α亚基几乎不能正常表达,在重组E. coli BL21(DE3) (pET32aNHBAX)中,腈水合酶活性仅为0.04U/mg。构建重组毕赤酵母表达质粒pPIC3.5kNHBAX,采用电穿孔转化法将其转入宿主菌P. pastoris GS115中,经过菌株培养和腈水合酶的诱导表达,筛选获得了优选菌株P. pastoris NH4。对P. pastoris NH4的细胞培养和腈水合酶的诱导表达条件进行优化,结果表明,重组腈水合酶在毕赤酵母中的表达水平可以达到0.52U/mg,但不能稳定积累。     

Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.     

转铁蛋白受体单链抗体与链亲和素融合基因的克隆及其在大肠杆菌中的可溶性表达

目的:转铁蛋白受体特异性富含于血脑屏障和肿瘤细胞的表面,是当前中枢神经系统疾病和肿瘤治疗中定向转运的重要靶标。拟获得在大肠杆菌中能高效可溶表达的转铁蛋白受体单链抗体与链亲和素(SA)的重组融合蛋白。方法:根据GenBank数据库报道的SA的核苷酸序列分段合成基因,连接后经PCR获得完整的基因片段,插入pGEM-T载体中测序。将序列正确的SA基因与大鼠转铁蛋白受体单链抗体基因ox26-scFv分别插入原核表达载体pTIG-Trx中,构建重组表达克隆pTIG-Trx/scFv-SA,并在大肠杆菌中诱导表达。ELISA检测融合蛋白的生物学活性。结果:对pGEM-T/SA克隆的测序结果显示,合成的SA基因与文献报道相符。重组融合蛋白在大肠杆菌中获得了可溶性表达,约占菌体上清总蛋白量的30%;ELISA结果表明该融合蛋白具备与转铁蛋白受体和生物素的结合的双重活性。结论:有活性的重组融合蛋白的获得为构建一个通用性的以转铁蛋白受体介导的血脑屏障和肿瘤转运靶向载体打下了基础。