Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.     

溶氧反馈分批补料高密度培养人骨形成蛋白-2工程菌

对表达人骨形成蛋白－2成熟肽的基因工程大肠杆菌E.coli DH5α/pDH-B2m在500mL摇瓶中进行了培养条件的摸索实验,并在此基础上扩大至NBS Bioflo IV20L发酵罐,利用溶氧反馈－分批补料培养技术：在培养过程中保持适当的溶解氧（40％）,以溶氧值在线反馈控制搅拌速度及流加补料培养基,使细菌保持适当的比生长率,成功地进行了工程菌的高密度培养,最终菌体密度达OD600＝57,每升干菌量22.8g,目的蛋白的表达量占细菌总蛋白的30％,人骨形成蛋白－2成熟肽的理论产率达到3.59g/L。     

Investigations on anti-Aspergillus properties of bacterial products

AIMS: To investigate the anti-Aspergillus properties of bacterial products. METHODS AND RESULTS: In the present study, 12 bacterial strains were screened for antifungal activity against Aspergilli. The culture supernatant and lysates of Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli (BL21, DH5alpha, HB101, XL Blue), Klebsiella pneumoniae, Streptomyces thermonitrificans, Streptococcus pneumoniae, Enterobacter aerogenes, Staphylococcus aureus and Salmonella typhi were examined for antifungal activity in protein concentration ranging from 1000.0 to 7.8 microg ml-1 using microbroth dilution assay. The lysate of Salm. typhi and E. coli BL21 exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 15.6-31.2 microg ml-1 by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, a concentration of 3.1 microg disc-1 of Salm. typhi lysate showed significant activity against Aspergilli. Escherichia coli BL21 exhibited similar activity at 6.2 microg disc-1. The work on identification of molecule endowed with antimycotic properties is in progress. CONCLUSION: The products of Salm. typhi and E. coli demonstrated significant activity against Aspergillus species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that E. coli has been reported for anti-Aspergillus activity. It could be an important source of biologically active compounds useful for developing better new antifungal drugs/or probiotics.     

Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301.

A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively.     

信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响

【目的】研究不同的信号肽和化学通透剂对重组环糊精葡萄糖基转移酶(CGTase)胞外分泌的影响,提高CGTase的胞外分泌量。【方法】扩增地芽孢杆菌CHB1(Geobacillus sp.CHB1)的CGTase基因,构建带有地芽孢杆菌CHB1自身信号肽、Omp A、Pel B信号肽和不带信号肽的4种重组质粒;比较4种重组质粒对重组CGTase胞外分泌的影响,筛选最优的信号肽;考察甘氨酸、Triton X-100、SDS和Tween 80四种化学通透剂对重组CGTase胞外分泌的影响,确定最佳的化学通透剂及其浓度。【结果】Omp A信号肽介导的分泌效果最好,胞外酶活达到7.44 U/m L,分别是Pel B、CHB1信号肽的2.04倍和11.27倍,不带信号肽的重组质粒菌胞外检测不到酶活;携带Omp A信号肽的重组质粒菌发酵48 h,同时添加浓度为0.6%的甘氨酸和0.3%的Triton X-100,胞外酶活达最大到14.27 U/m L;SDS和Tween 80对该酶的胞外分泌具有明显的抑制作用。【结论】Omp A信号肽的介导效果最佳,同时添加浓度为0.6%和0.3%的甘氨酸和Triton X-100可以有效促进胞外分泌,为该重组酶的高效胞外分泌提供了一种有效的方法。     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

Improving the growth rate of Escherichia coli DH5alpha at low temperature through engineering of GroEL/S chaperone system

GroEL/S is a molecular chaperone system in Escherichia coli which not only assists the folding of intracellular proteins but also affects the cellular activity against the change of environmental condition. Here we show that the growth rate of E. coli DH5alpha can be improved at low temperature by expressing a GroEL/S variant achieved through irrational protein engineering approach. The GroELS variant (GroELS(var)) accelerating the growth of E. coli DH5alpha was screened through enrichment culture of the mutant libraries obtained by random mutagenesis. E. coli DH5alpha harboring the groELS(var) gene exhibited approximately 1.5-2 times higher growth rate compared to the strain with wild-type GroELS at 15-30 degrees C. At 10 degrees C, a temperature that the growth of E. coli DH5alpha almost stops, the GroELS(var) triggered the growth of E. coli DH5alpha. We identified that seven nucleotides of groELS gene and six amino acids of the GroELS were changed through the mutagenesis and screening. Site directed mutagenic analysis revealed that H360 in GroEL(var) is the most crucial residue determining the activity of GroELS(var) and more than one of the other residues in GroEL(var) may be additionally involved in the activity of GroELS(var). The improvement of growth rate induced by the GroELS(var) was observed only in the strain DH5alpha and not detected in other E. coli strains, such as BL21, BW25113, codon+, JM110, Top10, and XL1-blue.     

假单胞菌株M18 gacA基因的克隆、表达及蛋白纯化

GacA是GacS/A双元调控系统的一个组分, 克隆假单胞菌株M18中的gacA基因。测序结果同Pseudomonas sp. PAO1比较, 该基因2个碱基的改变未引起氨基酸的改变。将该基因克隆到表达质粒pET28b (+)中, 将重组质粒经热激转化至大肠杆菌BL21 (DE3)中, IPTG诱导表达。表达产物经金属螯合层析柱纯化, 蛋白纯度约为98%, 质谱鉴定证明纯化的蛋白为GacA蛋白。CD谱分析GacA包含了4% α-helix, 48% β-sheet 和48%无规则卷曲。GacA蛋白的获得为进一步研究其晶体结构、生物学性能以及GacS/A双元调控系统奠定了基础。     

Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli

The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.     

西藏小型猪Leptin及其受体胞外区片段的克隆及原核表达

目的克隆及原核表达西藏小型猪瘦素（Leptin）成熟肽及瘦素受体胞外区片段。方法根据西藏小型猪瘦素序列（GenBank号：GQ240885.1）和猪瘦素受体基因胞外域序列（GenBank号：AF167719.1）分别设计并合成两对引物扩增瘦素、瘦素受体基因胞外域编码区1654-2319位片段,以西藏小型猪组织总RNA为模板,经反转录-聚合酶链反应（RT-PCR）方法获得了特异性片段。再以该两个特异性片段为模板,另外设计两对带有BanHⅠ和HidⅢ酶切位点的套式引物分别扩增瘦素64-504位（成熟肽编码区）和瘦素受体基因胞外域编码区1655-2314位的cDNA片段,将该两片段克隆入pMD18-T载体并转化感受态细菌E.coli DH5α测序并永久保存。此两片段经酶切后克隆到表达载体pRSET A的BamHⅠ和HindⅢ两酶切位点之间,构建重组质粒pR-OB和pR-OBR-a并在大肠杆菌E.coli BL21（DE3）中表达,SDS-PAGE电泳鉴定表达产物。结果在IPTG诱导下促使重组菌pR-OB表达了相对分子质量约18×103左右的融合蛋白;重组菌pR-OBR-a表达了相对分子质量约27×103左右的融合蛋白。结论说明重组质粒pR-OB、pR-OBR-a在大肠杆菌BL21（DE3）中分别可表达西藏小型猪瘦素成熟肽、瘦素受体片段蛋白,为进一步研究瘦素、瘦素受体功能和应用提供了基础。     

内毒素结合肽的原核表达、纯化及生物学活性鉴定

重组人内毒素结合肽 (endotoxinbindingpeptide ,EBP)融合蛋白在大肠杆菌中表达 ,分离和纯化后对其进行生物学活性观察 .将构建好的PinpointⅩa3 EBP生物素融合表达载体转化大肠杆菌DH5α ,IPTG诱导表达菌株 ,亲和层析法纯化表达产物 ,因子Ⅹa(factorⅩa)切割分离内毒素结合肽 ,采用凝胶过滤和反相液相高效色谱法两步纯化 ,从相对分子质量、N端 1 0个氨基酸的序列分析等方面进行鉴定 ;利用人单核细胞U937对重组内毒素结合肽进行了生物学活性的检测 .结果发现 ,内毒素结合肽以包涵体形式存在 ,因子Ⅹa酶切融合蛋白后得到 3 5kD的内毒素结合肽 ,纯化后内毒素结合肽纯度达 99%以上 ,N端 1 0个氨基酸的分析结果与预期相符 ;初步证实内毒素结合肽具有较好的LPS结合活性 ,能够抑制LPS的作用 .经原核表达及纯化复性 ,获得了具有较好生物学活性的内毒素结合肽 ,为进一步研究其功能奠定了良好的基础     

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.     

Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases.

A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5 alpha as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene.     

布鲁氏菌L7/L12蛋白的基因克隆与原核表达

本研究对羊布鲁氏菌L7/L12蛋白进行了表达和纯化。首先从布鲁氏菌M5基因组中克隆L7/L12目的基因片段,连接至pMD-19T载体,转化入E.coli DH5α感受态细胞,PCR鉴定及测序鉴定正确后对其进行双酶切,构建重组质粒pGEX-6P-1-L7/L12并利用E.coli BL21(DE3)进行诱导表达。羊布鲁氏菌L7/L12基因片段大小为375 bp。SDS-PAGE检测蛋白大小为13 kD,与预测值相符。Western blotting方法检测其免疫学特性。实验结果表明,成功构建了pGEX-6P-1-L7/L12原核表达载体,并在大肠杆菌中成功表达了L7/L12重组蛋白,Western blotting法检测其具有免疫反应。本实验为下一步研究蛋白功能及布鲁氏菌新型疫苗的研制提供了实验基础。     

抗人CD19单链抗体基因的构建、表达及功能测定

采用RT-PCR方法从分泌抗人类白细胞表面分化抗原CD19单克隆抗体的杂交瘤细胞中克隆出V_H和V_L可变区基因,再通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人CD19单链抗体（抗CD19-ScFv）基因。将其克隆至表达载体PET28a并在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明,抗CD19-ScFv在BL21（DE3）菌中获得表达,重组蛋白的相对分子量为27kD,表达产物以不溶性包涵体形式存在,经过溶解包涵体,镍柱亲和层析纯化和体外复性过程,获得了高纯度的单链抗体片段。流式细胞分析结果证实抗CD19ScFv可与人类白细胞表面的分化抗原CD19结合,保留了鼠源性单抗与CD19结合活性。抗人CD19-ScFv的构建与表达,为下一步针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础。     

东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因,以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列,设计引物,用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因,该基因开放阅读框架编码59个氨基酸残基,其中前22个为信号肽,成熟肽为37个氨基酸残基,经PCR扩增除去信号肽序列,克隆到pTreHisA质粒中,在E．coli中表达了分子质量为7ku左右融合蛋白,表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链,6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-,推断其为K^ 通道阻断肽,命名为KChTX1。已被Gene-bank收录,收录号为AY129234。     

Expression of human parathyroid hormone in Escherichia coli

Human parathyroid hormone (PTH) has been expressed in Escherichia coli as a cro-beta-galactosidase-hPTH fusion protein under temperature-sensitive control of the lambda phage PR promoter. The lacZ gene has been truncated to a different extent revealing an optimal length of the prokaryotic peptide portion between 199 and 407 amino acid residues. Up to 250 mg of pure fusion protein have been obtained from 1-liter E. coli culture by stepwise solubilization with urea. The linkage between the prokaryotic and the eukaryotic protein moiety consists of an Asp-Pro peptide bond and therefore is easily cleavable by acid treatment. A simple procedure for the purification of the hormone is described. The resulting recombinant hormone reacts with anti-PTH antibodies and stimulates renal adenylate cyclase identically to bovine or human PTH.     

蛙皮素-蜂毒素杂合肽基因在大肠杆菌中的克隆与诱导表达

抗菌肽 (Ａｎｔｉｂａｃｔｅｒｉａｌｐｅｐｔｉｄｅｓ)原指昆虫体内经诱导产生的一类分子量在 4ｋＤ左右 ,具有抗菌活性的碱性多肽物质[2 ] 。这类抗菌剂最初是从昆虫、哺乳动物、两栖动物等的防御系统中分离得到的。抗菌肽对革兰氏阳性及阴性细菌、病毒、原虫和发生病变的真核细胞等有杀伤作用 ,具有很强的广谱抑菌作用 ,但对正常哺乳动物细胞无杀伤作用[3 ] 。蛙皮素还具有抗肿瘤活性而没有溶血活性[4 ] ,虽然蜂毒素具有溶血活性 ,但其溶血功能区位于Ｃ端的亲水区域[5] ,两者的极性区域正好相反。据研究表明抗菌肽都会形成两亲螺旋结构的…     

温敏类弹性蛋白多肽的基因设计及重组克隆表达

温敏类弹性蛋白多肽(elastin-like polypeptides, ELPs)作为新型的药物载体,在肿瘤治疗中具有广阔的应用前景。本研究根据大肠杆菌密码子的偏好性和简并性,将高度连续重复氨基酸的多种遗传密码引入基因序列中,利用依次插入单体基因和递归定向连接技术,成功构建了以缬氨酸为客座氨基酸的ELPs基因的表达质粒库,即p ET28-ELP-V_5～pET28-ELP-V₅₀。将pET28-V₅₀转化至表达宿主Escherichia coli BL21(DE3)中,经重组菌的培养和IPTG诱导,SDS-PAGE结果显示ELP-V₅₀在大肠杆菌中成功获得了可溶性表达,与预期分子量大小一致。本研究为进一步构建不同分子量的ELPs基因库提供了新的思路,并为重组表达获得特定相变特性的多肽分子奠定了基础。