Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG

The Escherichia coli McrA protein, a putative C⁵-methylcytosine/C⁵-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA’s binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that N(Y > R) m5CGR is the canonical binding site for rMcrA-S. In addition to binding HpaII-methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind if A, C, T or U is placed across from the m5C residue, but does if I is opposite the m5C. These results provide the first systematic analysis of McrA’s in vitro binding specificity.     

High-level expression of the Arabidopsis thaliana ethylene receptor protein ETR1 in Escherichia coli and purification of the recombinant protein

Ethylene responses in plants are mediated by a small family of membrane integral receptors including the ETR1 gene product which are similar to the two-component bacterial histidine kinase regulators. Detailed biochemical and structural analysis of the ethylene-receptor family was hampered by the scarce amount of pure protein. Here, we report the construction, expression, and single-step purification of the ETR1 receptor protein from Arabidopsis thaliana in a bacterial expression system. The DNA fragment encoding the mature ETR1 receptor protein was subcloned into the pET15b expression vector and highly expressed in derivatives C41(DE3) and C43(DE3) of the Escherichia coli strain BL21(DE3). The recombinant protein was solubilised from the bacterial cells using mild non-denaturing detergents and purified to homogeneity by Ni-NTA affinity chromatography, yielding approximately 2-3 mg pure protein per litre of cells. The molecular mass of the purified protein was estimated to be 78 kDa by SDS-PAGE. The expression and purification of recombinant ETR1 reported here provide a basis for detailed functional and structural studies of the receptor protein, which might help to understand signal perception and signal transduction of the phytohormone ethylene on the molecular level.     

灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

以灵杆菌基因组DNA为模板,PCR扩增非特异性核酸酶 (Non-specific nuclease,NU) 基因,并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21,经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU,MBP-NU),其最佳诱导表达条件为37 ℃,0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性,在37 ℃、pH 8.0时活性最高,比活力为1.11×106 U/mg,目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid,EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride,PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响,因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。     

APOBEC3G蛋白的原核表达及纯化

目的：原核表达重组APOBEC3G蛋白,为其功能及免疫原性研究奠定基础。方法：提取H9细胞全细胞基因组RNA,通过RT-PCR获得目的基因,经纯化、酶切后克隆到原核表达载体pET32a中,转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,并对表达条件和纯化条件进行优化;利用Western Blot分析鉴定目的蛋白。结果：构建了APOBEC3G蛋白的原核表达载体Apo-His-pET32a,并在大肠杆菌中获得高表达,目的蛋白以可溶性蛋白形式存在;经Ni-NTA亲和层析柱一步纯化,获得了高纯度的重组APOBEC3G蛋白,蛋白浓度可达1．2mg／mL;Westem Blot显示获得了目的蛋白。结论：在原核表达系统中表达、纯化了可溶性APOBEC3G蛋白,为进一步对其进行免疫原性和功能研究奠定了基础。     

Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli

The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-beta-D-galactoside (IPTG) was found to be 5 microM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degrees C, respectively. The Km and Vmax are 17.6 microM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.     

KDR酪氨酸激酶的克隆表达及纯化

VEGF作为一种血管内皮细胞有丝分裂原,通过与内皮细胞表面特定受体结合,从而导致新生血管的形成,其中VEGFR-2(KDR/FIk-1)在肿瘤血管形成中起重要作用,因此其抑制剂有可能发展成为治疗肿瘤的新药.采用大肠杆菌成功表达了具有酪氨酸激酶活性的KDR酪氨酸激酶催化域(KDR-CD).采用RT-PCR从人脐静脉内皮细胞中提取总RNA,获得KDR-CD的编码基因,将其克隆至pET-30a载体,在E. coli BL21(DE3)中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化,Western blotting结果显示表迭的KDR-CD蛋白自身磷酸化,重组KDR-CD蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性.     

Molecular cloning and sequencing of mcrA locus and identification of McrA protein inEscherichia coli

The Mcr systems (previously known as Rgl systems) ofEscherichia coli recognize and cleave specific sequences carrying methylated or hydroxymethylated cytosines. We have cloned the mcrA gene and determined its nucleotide sequence. An 831 base pair sequence encodes the McrA protein. Analysis of the sequence data reveals that there arc additional ORFs internal to the above. A phage T7 expression system was used to determine the protein products encoded by the cloned mcrA gene. The results clearly show that a 31 kDa polypeptide is responsible for McrA activity. This is in agreement with the molecular weight deduced from sequence data. McrA protein was found to be localized in the outer membrane ofEscherichia coli. To our knowledge this is the first restriction enzyme localized in the outer membraneof Escherichia coli. Presented in part at the Second New England Biolabs Workshop on Biological DNA Modification, September, 1990, Berlin     

炭疽芽孢杆菌EA1蛋白的融合表达和纯化

目的：原核表达重组炭疽芽孢杆菌EA1蛋白。方法：用PCR方法从炭疽芽孢杆菌A16R疫苗株染色体中扩增编码EA1蛋白的eag基因序列,经过纯化、酶切后克隆到含有GST标签的原核表达载体pGEX-6P-2中,构建重组载体pGEX-EA1;将空载体（作为对照）、重组载体转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,对其表达和纯化条件进行优化;利用Western印迹检测融合蛋白的表达。结果：构建了EA1蛋白的融合表达载体,并在大肠杆菌中获得高效表达;经Glutathione Sepharose 4B纯化获得了EA1蛋白;Western印迹表明,此蛋白可与GST标签抗体反应。结论：在原核表达系统中表达并纯化得到EA1融合蛋白,为进一步对其进行功能研究奠定了基础。     

串联亲和纯化原核表达载体的构建

【目的】构建串联亲和纯化原核表达载体,用于研究细菌中(生理状态或接近生理条件下的)蛋白-蛋白相互作用。【方法】设计并合成两条串联亲和标签序列,分别可以在靶蛋白N端和C端融合Protein G和链亲和素结合肽(Streptavidin binding peptide,SBP)标签;以pUC18载体为骨架,去除原有的阻遏蛋白基因,构建组成型表达载体pNTAP和pCTAP。【结果】成功构建N端和C端标签表达载体pNTAP和pCTAP,它们在大肠杆菌(Escherichia coli)BL21(DE3)、肠出血性大肠杆菌O157:H7和痢疾杆菌福氏5型M90T菌株中都可以实现表达。【结论】本实验构建的两个串联亲和纯化表达载体可以在部分革兰氏阴性细菌中表达,为研究细菌内蛋白-蛋白相互作用及致病菌毒力蛋白的作用机制奠定了基础。     

重组斑马鱼 p8蛋白的原核表达及纯化

目的：在大肠杆菌中重组表达斑马鱼p8蛋白并纯化。方法：PCR扩增斑马鱼p8蛋白基因编码区,连接到带有6×His标签的原核表达载体pET-28a中,构建重组表达质粒pET-28a-p8并转化大肠杆菌BL21（DE3）,用IPTG诱导表达;优化表达条件后用Ni^2＋柱纯化重组蛋白。结果：构建了pET-28a-p8重组质粒;目的蛋白在大肠杆菌中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的12．8×10^3。结论：获得了斑马鱼p8融合蛋白,为其生物学功能研究奠定了基础。     

人工合成草鱼生长激素cDNA在大肠杆菌中的表达

经密码子优化的人工合成草鱼生长激素cDNA与表达载体pET-28a( )重组,构建重组表达质粒PET－GH。转化在肠杆菌BL21（DE3）,筛选阳性克隆,IPTG诱导表达,12．5％的SDS－PAGE分析显示,大肠杆菌表达产物中含有与草鱼生长激素分子量一致的新增蛋白带,激光密度扫描,其产量约占菌体总蛋白的40％,金属离子螯合层析柱亲和纯化,获得电泳纯的重组蛋白。Western-blotting和酶联免疫吸附受体法检测证实,重组蛋白与抗草鱼生长激素的多克隆抗体发生特异性结合;复性合的重组蛋白有与天然草鱼生长激素一致的生物学活性。     

Over-expression, purification, and characterization of recombinant NAD-malic enzyme from Escherichia coli K12

NAD(+)-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E. coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombinant NAD-ME could be produced mainly in a soluble form. The monomeric molecular weight of recombinant NAD-ME was about 65 kDa, whereas monomer, homotetramer, and homooctamer were formed in solution as revealed by nondenaturing polyacrylamide gel electrophoresis analysis. Finally, the K(m) values of NAD-ME for L-malate and NAD were determined as 0.420+/-0.174 and 0.097+/-0.038 mM, respectively, at pH 7.2. By using this over-expression and purification system, recombinant E. coli K12 NAD-ME can now be obtained in large quantity necessary for further biochemical characterization and applications.     

酿酒酵母ATP-硫酸化酶在大肠杆菌中的表达纯化及其在焦测序中的应用

ATP-硫酸化酶(ATPS,EC2.7.7.4)是一种可逆催化ATP和SO42-反应生成腺嘌呤-5′-磷酸硫酸(APS)和焦磷酸盐(PPi)的酶,已经用于焦测序反应。以酿酒酵母(Saccharomyces cerevisias,CICC1202)基因组DNA为模板,用PCR扩增得到ATPS基因,并克隆到原核表达质粒pET28a( ),得到重组表达质粒pET28a( )-ATPS,在IPTG诱导下,携带pET28a( )-ATPS的大肠杆菌BL21(DE3)表达分子量约为60kD的带有His标签的ATPS酶,经镍亲和层析和超滤两步纯化后,可得到电泳纯级ATPS,比活达5.1×104u/mg,并成功应用于焦测序反应中。     

小鼠Nanog基因的克隆及其在大肠杆菌中的表达

按照nanog基因编码序列设计合成引物,利用RT-PCB从小鼠的囊胚期胚胎中扩增得到该 基因,并将该基因克隆到pET-28b(+)载体上,获得pET-28b(+)-nanog原核表达重组质粒,限制 性酶分析和DNA序列测定均证实该克隆插入片段为nanog基因编码序列。重组质粒转化大肠杆 菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得了高效表达,western杂交证实该 蛋白具有6-His抗原活性,从而证实目的蛋白为Nanog蛋白。     

虾夷扇贝过敏原tropomyosin的克隆表达、纯化及免疫学鉴定

从虾夷扇贝(Patinopecten yessoensis)肌肉中提取总RNA,RT-PCR克隆虾夷扇贝中变应原原肌球蛋白的全长基因,根据序列设计带有酶切位点的特异性引物,扩增扇贝tropomyosin的完整开放阅读框,与pET-28a载体连接并转化大肠杆菌Escherichia.coli BL21(DE3),诱导表达后,Ni2+亲和层析柱纯化重组蛋白,Western-blot检测其免疫学活性。经序列测定,该基因含有长度为855bp的开放阅读框,编码284个氨基酸,其在GenBank数据库中的登录号为EU839640。SDS-PAGE检测该重组变应原在大肠杆菌中高效表达36kD的目的蛋白,且重组变应原具有良好的IgE结合活性。研究获得了具有变应原活性的重组虾夷扇贝tropomyosin,为扇贝过敏性疾病的诊断和治疗奠定了基础。       

重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框（ORF）插入原核表达载体pGEX6P1 GST基因下游的多克隆位点（MCS）.将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.     

Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli McrA endonuclease

McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates. We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system. We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein. Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified lambda and T4gt, respectively) and for induction of the E. coli SOS response in the presence of M.HpaII methylation, indicative of DNA damage. Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a linker region largely tolerant of amino acid insertions. DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4gt. Disruption of the N-terminal domain abolishes restriction of both substrates. Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified lambda in vivo. We suggest a common explanation for this "restriction without damage" and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions. Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the lambda genome disrupts the phage development program at an early stage. We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict lambda but did not restrict T4gt at all. This mutant may have a selective impairment in substrate recognition, distinguishing methylated from hydroxymethylated substrates. The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.     

重组人脑源性神经营养因子在大肠杆菌中的可溶性表达及纯化

按照人脑源性神经营养因子(hBDNF)基因成熟肽编码序列设计合成引物,从人基因组DNA中扩增出360bp的片段,插入到改构载体pTIG-trx上,获得了pTIG-trx—BDNF原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为hBDNF基因成熟肽编码序列。将该重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得了高效可溶表达,并对表达产物进行了分离纯化,得到纯度大于83％的样品,Western杂交证实该蛋白具有hBDNF抗原活性。     

克雷伯杆菌甘油脱氢酶基因的克隆表达与纯化

以克雷伯杆菌(Klebsiella pneumoniae)基因组DNA为模板, 运用PCR扩增得到编码甘油脱氢酶(GDH)的基因(gldA), 并克隆到pMD-18T载体上, 构建克隆载体pMD-gldA。经测序正确后, 将gldA亚克隆至表达载体pET-32a(+)上构建表达质粒pET-32gldA。在乳糖诱导下, 携带pET-32gldA的E. coli BL21 (DE3)高效表达分子量约为54 kD的可溶性蛋白。表达产物带有His6-tag标记, 选用Ni柱对表达产物进行纯化, 纯化后酶液的比活为188 u/mg, 纯化倍数和回收率分别为3倍和67.5%。