大肠杆菌发酵生产重组人颗粒溶素

目的:在FUS-50L生物反应器中利用补料分批培养技术培养表达含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS株,生产重组颗粒溶素(GNLY)。方法:选取含pET28a( )-GNLY的BL21(DE3)pLysS菌株单个克隆分级培养,将制备的二级种子液接种于发酵罐中。在发酵过程中,控制溶氧为30%～50%,温度为37℃;在基础培养基内生长4h后,补加以甘油为碳源的补料,继续生长到11h;加入葡萄糖至终浓度为1%,30℃诱导表达6h;收集菌体,纯化制备目的蛋白。利用Western印迹检测重组蛋白的抗原性,用CFU方法检测其生物学活性。结果:发酵液中最终菌体密度达80g/L;纯化所得重组蛋白约占菌体总蛋白的5%,含量为60mg/L;经鉴定所获重组颗粒溶素有较好的免疫学活性和生物学活性。结论:用含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS表达系统,可得到具有生物活性的重组颗粒溶素,为大批量生产提供了条件。     

抗菌肽GK1在大肠杆菌中的融合表达

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。     

猪尿酸氧化酶在大肠杆菌中的表达、纯化与部分酶学性质分析

本研究报道了猪尿酸氧化酶(Porcine urate oxidase,pUOX)的原核表达载体的构建、pUOX的蛋白表达条件的优化以及对pUOX经纯化后进行活性检测和酶学性质分析。利用RT-PCR从猪肝总RNA中克隆pUOX,定向插入原核表达载体pET30a(+)中,构建表达载体pET30a(+)/pUOX,并转化到大肠杆菌(Escherichia coli)BL21(DE3)中。重组质粒pET30a(+)/pUOX经双酶切鉴定和序列分析,证实已成功构建了重组表达载体。重组表达菌经IPTG诱导表达了约为41kD的蛋白,与预期分子量一致,并对pUOX蛋白表达条件进行了优化,表达的蛋白主要以包涵体的形式存在于细胞中,包涵体经过变性、复性后,用Ni2+-NTA对复性蛋白进行亲和纯化,并对纯化蛋白进行了活性检测和酶学性质分析,纯化的重组pUOX的比活为50.63IU/mg,并发现重组蛋白在最佳温度、热稳定性等方面与天然pUOX相同,为后续动物实验奠定重要的基础。     

羊口疮病毒蛋白ORFV035的表达、纯化和多克隆抗体的制备

克隆表达羊口疮病毒蛋白ORFV035,并制备其多克隆抗体,为后续对病毒复制、装配、形态发生和成熟过程的研究奠定基础。PCR扩增羊口疮病毒ORFV035基因,将其与质粒pET-30a(+)经Bam HⅠ和HindⅢ双酶切后连接,构建重组质粒pET30a-035。重组质粒经双酶切和测序鉴定,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,纯化后目的蛋白免疫小鼠,制备多克隆抗体并对其进行鉴定。成功构建了重组质粒pET30a-035,在大肠杆菌BL21中以包涵体形式高效表达。包涵体洗涤、溶解后进行Ni柱纯化,得到纯度较高的ORFV035-his融合蛋白。以纯化蛋白免疫小鼠获得多克隆抗体。Western blot检测显示该多抗可以识别天然ORFV035蛋白。成功诱导表达、纯化ORFV035蛋白并制备ORFV035多克隆抗体。     

鲎血细胞中脂多糖结合蛋白TALF在大肠杆菌中的表达研究

TALF(Tachyleus antilipoposaccharide factor)对细菌内毒素（LPS）的核心部分有抑制作用。研究TALF cDNA基因在大肠杆菌中的表达,首先将TALF cDNA基因分别插入大肠杆菌表达载体pGEX-4T-2、pET22b、pET28a中,构建重组表达质粒,转化于大肠杆菌BL21(DE3)。结果表明克隆于pET22b、pET28a中的TALF cDNA基因没有表达,而融合了GST的TALF基因（GST-TALF）能够在大肠杆菌中表达,并形成包涵体。从1L培养基中可获得4mg纯度为91%的GST-TALF融合蛋白。经复性和纯化后的融合蛋白GST-TALF几乎检测不到抑菌活性及LPS中和活性,但该融合蛋白经凝血酶消化后表现出明显的体外抑菌活性及LPS中和活性。     

结核分枝杆菌Rv3369基因的表达和纯化

在大肠杆菌中高效表达结核分枝杆菌Rv3369蛋白,获得纯化的重组蛋白rRv3369。通过聚合酶链反应(Polymerase chain reaction,PCR)扩增Rv3369基因;以质粒pET28a为表达载体,构建重组质粒,转化大肠杆菌BL21(DE3);以异丙基硫代半乳糖苷(IPTG)诱导表达目的蛋白,通过SDS-PAGE鉴定rRv3369在大肠杆菌中的表达,确定rRv3369在大肠杆菌中的表达形式;采用Ni-NTA His.Bind Resin来纯化重组蛋白。重组质粒pET28a-Rv3369中目的基因测序结果与报道序列相同。分子量约19.5kDa,表达量约占菌体总蛋白的18%,纯化后的重组蛋白样品经SDS-PAGE和激光密度扫描分析表明其纯度为90%以上,每100mL培养菌可获得1.56mg左右的重组蛋白。用亲和层析法纯化的重组蛋白纯度较好。     

Flt3配体胞外域的原核表达纯化及活性鉴定

目的:构建pET32a(+)-hFLext原核表达载体,诱导hFLext蛋白表达、纯化及活性鉴定.方法:以人淋巴细胞cDNA文库为模板,克隆hFlext,构建pET32a(+)-hFLext重组表达载体.转化大肠杆菌BL21,IPTG诱导蛋白表达,镍珠亲合层析纯化蛋白,SDS-PAGE及Western blot鉴定.细胞增殖实验检测其生物学活性.结果:成功克隆获得hFLext,并构建了pET32a(+)-hFLext重组表达载体.在大肠杆菌BL21,经1 mM IPTG 30℃诱导12 h,成功表达Trx-hFLext融合蛋白,主要以包涵体形式存在.经8M尿素变性包涵体蛋白,逐步透析复性,镍珠亲合层析纯化蛋白,SDS-PAGE及Western blot鉴定,成功获得高纯度的Trx-hFLext融合蛋白.细胞增殖实验证实其具有生物学活性,能够有效刺激脐血细胞增殖.结论:成功构建了pET32a(+)-hFLext重组表达载体,表达、纯化了具有生物学活性的Trx-hFLext融合蛋白,为造血干/祖细胞的体外扩增研究奠定了基础.     

柑桔溃疡病菌重组单链抗体的高效表达及活性检测

运用PCR方法扩增利用核糖体展示技术筛选的抗柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,XAC)的单链抗体(ScFv95)基因片段,将单链抗体基因重组到原核表达载体pET30a( )中,构建单链抗体高效表达载体pET30a( )-XAC-ScFv。再将pET30a( )-XAC-ScFv质粒转化进大肠杆菌BL21(DE3)后诱导表达,并对表达产物进行纯化、复性及活性检测。获得了抗XAC单链抗体的高效表达蛋白,以包涵体形式存在的表达蛋白大小约32kDa。包涵体蛋白经过变性、纯化和复性后,初步获得有功能的单链抗体。同时用Biacore分析XAC-ScFv-95与XAC LPS作用,结果表明复性后的XAC-ScFv-95具有较高的亲和力,从而为柑桔溃疡病菌XAC的免疫诊断和防治研究提供了新的工具和途径。     

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。     

破骨细胞形成抑制因子TNFR结构区在大肠杆菌中表达、抗体制备及活性测定

破骨细胞形成抑制因子(OPG)对骨的重建与再吸收有重要调节作用,其TNFR结构区行使抑制破骨细胞形成与活性的功能。通过PCR将该区基因片段克隆出来,插入表达载体PET-28a质粒多克隆位点,重组质粒转入大肠杆菌BL21中进行表达,表达产物以包涵体形式存在,包涵体经变性复性后,亲合层析获得重组蛋白。纯化的产物作为抗原免疫兔,得到较高特异性的兔源多克隆抗体。利用小鼠降血钙实验检测变性复性后产物的活性,结果表明该重组蛋白有一定的生物活性。     

Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.     

High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system

BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.     

Expression, purification, and functional characterization of recombinant human interleukin-7

Human interleukin-7 (IL-7) is a member of the interleukin family. Numerous studies have demonstrated IL-7's effect on B- and T-cell development as well as its potential in various clinical applications. Previously, a study reported that IL-7 could be purified from inclusion bodies using a prokaryotic system, however, the required refolding step limits the recovery rate. This study was designed to produce a bioactive recombinant human IL-7 (rhIL-7) in a eukaryotic expression system in order to obtain higher yields of the protein with simpler purification steps. We cloned human IL-7 cDNA and successfully expressed active recombinant protein in yeast using the Pichia pastoris expression system. A simple purification strategy was established to purify the rhIL-7 from the fermentation supernatant, yielding 35 mg/L at 95% purity by the use of a common SP Sepharose FF cation-exchange chromatography. Functional analysis of the purified rhIL-7 by the pre-B cell MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) proliferation assay demonstrated a specific activity comparable to commercial sources. These results suggest that purification of rhIL-7 from yeast provides a sound strategy for large-scale production of the rhIL-7 for clinical applications as well as basic researches.     

Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form

(MTG) The Streptoverticillium transglutaminase gene, synthesized previously for yeast expression, was modified and resynthesized for overexpression in E. coli. A high-level expression plasmid, pUCTRPMTG-02(+), was constructed. Furthermore, to eliminate the N-terminal methionine, pUCTRPMTGD2 was constructed. Cultivation of E. coli transformed with pUCTRPMTG02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200-300 mg/liter) as insoluble inclusion bodies. The N-terminal amino acid residue of the expressed protein was methionine or serine (the second amino acid residue of the mature MTG sequence), respectively. Transformed E. coli cells were disrupted, and collected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTG restored the enzymatic activity. Refolded MTG, purified by ion-exchange chromatography, which had an N-terminal methionine or serine residue, showed activity equivalent to that of native MTG. These results indicated that recombinant MTG could be produced efficiently in E. coli.     

人β2-微球蛋白基因克隆及其在大肠杆菌中的高效表达

β2-微球蛋白(β2m)是主要组织相容性复合体(MHC)Ⅰ类分子的轻链部分,为制备MHCⅠ类分子四聚体的必要成分。根据已报道的序列设计特异引物,利用RT-PCR方法从人白细胞中克隆了β2m基因,并构建了成熟β2m的原核表达载体,在大肠杆菌中得到高效表达。表达的β2m大部分在包涵体中,经洗涤、变性和复性,并以强阴离子交换柱层析纯化,获得SDS-PAGE纯的人重组β2m,Western印迹法分析表明该蛋白具有与抗人天然β2m抗体反应的特性。此工作为制备MHCⅠ类分子四聚体奠定基础。     

Purification of bovine S100A12 from recombinant Escherichia coli.

S100A12, a member of the S100 family of EF-hand calcium-binding proteins, was purified from Escherichia coli cells expressing the corresponding cDNA. The procedure involved washing induced E. coli cells with EDTA-containing hypotonic solution, ion-exchange chromatography, and HPLC. Recombinant S100A12 was purified to homogeneity with the final yield around 6.7 mg per 20 ml of culture. The purified protein was identical to native S100A12 in the N-terminal amino acid sequence, lysylendopeptidase peptide mapping, mass spectrum, and Ca2+-dependent binding affinity to amlexanox, an antiallergy drug. However, the N-terminal methionine residue of the purified protein was not cleaved off as in the native protein. The method used in the present study permits the purification of recombinant S100A12 in large quantities and may also be applicable to preparation of other S100 family proteins.     

粒细胞集落刺激因子(G-CSF)在大肠杆菌中的高效表达及快速纯化工艺的建立

基因工程重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）主要用于癌症患者化疗后的粒细胞减少症．在正确克隆人Ｇ－ＣＳＦｃＤＮＡ的基础上,重点对Ｇ－ＣＳＦｃＤＮＡ的５′端进行了较为彻底的修饰,修饰后的基因插入ｐＢＶ２２０载体组建成功ｐＢＶ２２０／Ｇ－ＣＳＦ／２－１７４高效表达载体．表达后ＳＤＳ－ＰＡＧＥ分析其表达最高达５０％以上．根据Ｇ－ＣＳＦ表达形成包涵体这一特性,建立了一条简便、稳定,适用于大规模生产的分离纯化工艺流程．首先分离纯化包涵体,８ｍｏｌ／Ｌ尿素裂解包涵体,稀释复性蛋白,之后一步ＳＰ－ＳｅｐｈａｒｏｓｅＦＦ柱层析至均质．纯化的Ｇ－ＣＳＦ比活性达３．４×１０８Ｕ／ｍｇ蛋白,每升表达菌液回收的Ｇ－ＣＳＦ总活性达１．０６×１０１１Ｕ．纯化产物的Ｎ－端氨基酸序列分析表明,对甲硫氨酸的去除彻底,用于人体时可能具有较小的免疫原性和毒性     

High-level production and purification of Escherichia coli N-acetylneuraminic acid aldolase (EC 4.1.3.3).

The Escherichia coli gene which encodes N-acetylneuraminic acid aldolase was isolated by the polymerase chain reaction, cloned into the inducible expression vector pTTQ18, and overexpressed in E. coli. The high yield of aldolase was achieved through both optimum growth of cells and efficient expression of the aldolase gene (20-30% soluble cellular protein). The recombinant enzyme was purified to homogeneity with an activity of 1.2-2.2 U/mg, which compared favorably with that of commercial preparations of E. coli aldolase (1.1 U/mg) and Clostridium perfringens aldolase (0.4 U/mg). The cloning strategy, fermentation conditions, purification protocol, and activity assay are described.     

Human hemoglobin expression in Escherichia coli: importance of optimal codon usage.

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. alpha-Globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains. N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue. Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.     

Efficient production of native, biologically active human cystatin C by Escherichia coli

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the lambda PR promoter, an optimized Shine-Dalgarno sequence and the lambda cI 857 repressor. When induced at 42 degrees C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.