Over-expression in E. coli and purification of the human OCTN1 transport protein

The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6 h of induction with IPTG at 28 °C. The expressed protein with an apparent molecular mass of 54 kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6 h of induction with IPTG at 28 °C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3 M urea and applied to a Ni²⁺-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50 mM imidazole, 0.1% Triton X-100 and 50 mM 2-mercaptoethanol. A yield of about 3 mg purified protein per liter of cell culture was obtained.     

Over-Expression in <Emphasis Type="Italic">E. coli</Emphasis> and Purification of the Human OCTN2 Transport Protein

The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(+) carrying the glutathione S-transferase (GST) gene. The construct pET-41a(+)–hOCTN2 was used to express the GST–hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST–hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubilization with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni²⁺-chelating chromatography column. The purified GST–hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 protein. hOCTN2(R2K) cDNA was cloned in pET-21a(+) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni²⁺-chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure.     

Strategies of Bacterial Over Expression of Membrane Transporters Relevant in Human Health: The Successful Case of the Three Members of OCTN Subfamily

The OCTN subfamily includes OCTN1, 2, and 3 which are structurally and functionally related. These transporters are involved in maintenance of the carnitine homeostasis, which is essential in mammals for fatty acid β-oxidation, VLDL assembly, post-translational modifications, and other essential functions. Indeed, defects of these transporters lead to severe pathologies. OCTN1 and OCTN2 are expressed in many human tissues, while OCTN3 gene has been identified only in mouse and rat. The transporters mediate transport of carnitine and other substrates with different efficiencies and mechanisms. In order to over express the three proteins, a screening of many combinations of E. coli strains with plasmid constructs has been conducted. Only Rosetta(DE3) or Rosettagami2(DE3) gave significant expression. Higher protein amounts were firstly obtained with pET-41a(+) or pGEX-4T1 carrying fusion protein tags which required additional purification passages. Vectors carrying only a 6His tag, suitable for single passage purification, were preferred even though they lead to lower initial expression levels. Expressions were then increased optimizing several critical parameters. hOCTN1 was obtained with pH6EX3 in RosettaGami2(DE3)pLysS. hOCTN2 and mOCTN3 were obtained using pET-21a(+) in Rosetta(DE3). In particular, hOCTN2 was expressed only after codon bias, substituting the second triplet CGG with AAA (R2K mutant). The best growth conditions for hOCTN1 and mOCTN3 were 28 °C and 6 h of induction, while 4 h of induction for hOCTN2R2K. The proteins collected in the insoluble fraction of cell lysates, solubilized with sarkosyl, were purified by Ni-chelating chromatography. Final yield was 2.0, 3.0, or 3.5 mg/l of cell culture for mOCTN3, hOCTN1, or hOCTN2R2K. The data indicated that, in spite of the close evolutionary relations, several factors play different critical roles in bacterial expression of the three proteins, thus general criteria cannot be underlined. However, the strategy of dealing with related proteins revealed to be finally successful for over expressing all the three subfamily members.     

Expression and Activity Analysis of Fructosyltransferase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The fructosyltransferase gene was isolated and cloned from Aspergillus oryzae. The gene was 1368 bp, which encoded a protein of 455 amino acids. To analyze the activity of the expressed fructosyltransferase, the pET32a-fructosyltransferase recombined plasmid was transformed into Escherichia coli BL21. The fructosyltransferase gene was successfully expressed by Isopropyl-β-d-thiogalactoside (IPTG) induction. The molecular weight of the expression protein was about 45 kDa. The optimal conditions of protein expression were 25?°C, 0.1 mM IPTG, and 8 h of inducing time. The optimal concentration of urea dealing with inclusion body was 2.5 M. The expressed protein exhibited a strong fructosyl transfer activity. These results showed that the expressed fructosyltransferas owned transferase activity, and could catalyze the synthesis of sucrose-6-acetate.     

Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta?(DE3)pLysS by induction with 1 mM of isopropyl-β-d-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH?6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.     

抗汉坦病毒NP scFv基因表达及生物活性分析

诱导已构建的重组质粒pGEX-6P—1—scFv原核表达抗汉坦病毒核衣壳蛋白单链抗体,并用酶免疫实验检测单链抗体生物活性。用IPTG诱导重组原核表达质粒pGEX-6P-1-scFv表达抗汉坦病毒NP单链抗体融合蛋白,经亲和层析纯化,并应用SDS—PAGE电泳检测单链抗体融合蛋白,应用酶免疫实验检测抗NP单链抗体生物学活性。SDS—PAGE电泳检测显示,原核重组质粒pGEX-6P-1-scFv已表达分子量约为56ku的单链抗体融合蛋白;酶免疫实验检测显示,单链抗体具有与汉坦病毒NP抗原特异性结合的生物学活性。结果表明,已构建的原核表达重组质粒pGEX-6P-1-scFv,能够成功表达具有与汉坦病毒NP抗原特异性结合生物学活性的单链抗体。     

RING box protein-1 gene involved in flagellar disassembly of <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Ring box protein-1 (RBX1), also called Regulator of Cullins-1 (ROC1), is a key component of SCF (Skp-1, cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting protein substrates for degradation. Although RBX1 plays an important role in ubiquitination machinery of both prokaryotes and eukaryotes, studies on the RBX1 have not been involved in the unicellular green alga Dunaliella salina. In this study, a full-length RBX1 cDNA fragment of 817 bp was cloned using rapid amplification of cDNA end (RACE) technique. The full-length sequence contained an open reading frame of 411 bp encoding 136 amino acids. The predicted protein had a molecular molar mass of 14.8 kDa and pI of 5.9 with a high degree of homology to RBX1 from Chlamydomonas reinhardtii (92 %). Recombinant RBX1 was expressed in Escherichia coli BL21 and was purified and characterized. The apparent molecular mass of the recombinant protein was approximately 17 kDa, and the optimal induction time and concentration were 3 h and 0.1 mmol/L IPTG, respectively. The predicted 3D structures of RBX1 proteins contained RING-H2 finger domain including “Cys59-X2-Cys62-X30-Cys93-X1-His95-X2-His98-X2-Cys101-X10-Cys112-X2-Cys115.” The expression of RBX1 protein was increased by 132 % during flagellar disassembly and decreased by 76 % during flagellar assembly of D. salina. The expression of RBX1 mRNA had a similar tendency with the expression of RBX1 protein. The results indicated that RBX1 responded to flagellar disassembly of D. salina.     

Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E. coli

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. The goal of this study was to clone, efficiently express and purify the ecdysteroid-regulated protein (ERP) in the form of a GST fusion protein. The mature peptide-coding cDNA fragment was extracted from Chinese mitten crap (Eriocheir sinensis), and then after using PCR to obtain the open reading frame, a recombinant plasmid designated pGEX-4T-1_ERP was successfully generated and showed to efficiently express the ERP fusion protein as determined by SDS-PAGE. The resulting expressed protein was successfully purified by a combination of affinity and conventional chromatographic methods. After purification, the recombinant protein showed the expected size of 41 kDa on SDS-PAGE gels which was further confirmed by mass spectrometry and western blotting. Purification of recombinant protein was achieved by fast protein liquid chromatography. About 2.4 mg/l recombinant protein with purity more than 80 % was obtained.     

青岛文昌鱼S-腺苷高半胱氨酸水解酶基因表达载体的构建及表达纯化

为了在原核细胞中表达青岛文昌鱼Branchiostoma belcheri tsingtaunese S-腺苷高半胱氨酸水解酶(S-adeno-sylhomocysteine hydrolase,SAHH),采取构建文昌鱼SAHH基因的原核表达重组质粒pGEX-6P-1-SAHH的方法,转化入大肠杆菌JMl09感受态细胞中,IPTG诱导蛋白表达,并进行分离纯化.结果经SDS-PAGE分析,重组质粒在JM109中表达并纯化得到的融合蛋白大约为70 kDa,成功构建了文昌鱼SAHH基因原核表达载体,且重组载体表达出融合蛋白,分离纯化得到目的蛋白.     

白喉毒素无毒突变体CRM197基因的克隆与表达

目的构建白喉毒素(Diphtheria toxin)无毒突变体CRM197(Cross-reacting materials 197)的原核表达载体,并在大肠杆菌中表达重组蛋白。方法以白喉杆菌(ATCC39255)基因组DNA为模版,采用聚合酶链式反应(Polymerase chain reaction,PCR)扩增CRM197基因,插入表达载体pET11b中,构建重组原核表达质粒pET11b-CRM197。经双酶切及测序鉴定正确后,重组质粒被转化入大肠杆菌Rosetta 2(DE3)pLysS,IPTG诱导表达,表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒经双酶切及测序鉴定,结果表明与预期一致;表达的重组蛋白相对分子质量约58 000,并可与鼠抗CRM197单克隆抗体特异性结合。结论已成功构建了重组原核表达载体pET11b-CRM197,重组的CRM197蛋白在大肠杆菌中得到了表达,为以该重组突变体作蛋白载体制备结合疫苗奠定了基础。     

Overexpression in E. coli and Purification of the L. pneumophila Lpp2981 Protein

The Lpp2981 gene from Legionella pneumophila, the causative agent of Legionnaire’s disease, was cloned into the pMWT7 plasmid. The construct was used to express this gene in Escherichia coli. Five different bacterial strains were tested to overexpress the gene but without success. Sequence analysis revealed a cluster of four rare codons near the 5′-end of the gene. These codons were replaced with those commonly used in E. coli. The mutated Lpp2981 gene was successfully expressed in all the E. coli strains tested. The expressed protein (with an apparent molecular mass of 30 kDa) was collected in the insoluble fraction of the cell lysate, purified as inclusion bodies and functionally reconstituted into liposomes. The highest level of overexpression was obtained in E. coli C0214 after 6 h of induction with isopropyl-β-d-thiogalactopyranoside at 37 °C, yielding 74 mg of purified protein per liter of culture. We conclude that the clustering of rare codons at the 5′-end of the open-reading frame is a critical factor for the heterologous expression of Lpp2981 in E. coli.     

Cloning,expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-d-galactopyranoside but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K _m and V _max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and ¹³C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.     

Cloning, Expression, and Characterization of Iron Superoxide Dismutase in Sonneratia alba, a Highly Salt Tolerant Mangrove Tree

In this study, a novel iron superoxide dismutase (FeSOD) gene from Sonneratia alba was cloned and then expressed in Escherichia coli Rosetta-gami, designated as SaFeSOD. The DNA sequence of SaFeSOD contained a 786-bp open reading frame which encodes a 261 amino-acid protein of 30.0 kDa. The 651-bp fragment coding for putative mature SaFeSOD was amplified and inserted into pET15b for expression. This recombinant SaFeSOD was subsequently isolated by Ni-trap column protein purification system. The apparent molecular mass of the purified enzyme was 25 kDa on SDS-PAGE. In comparison with FeSODs from other plant species, all iron-binding sites (His 27, His 80, Asp 164 and His 168) of SaFeSOD were conserved. SaFeSOD was found to have good pH stability in the pH range of 3.5–9.5 at 25 °C after 1 h incubation and was relatively stable and showed 78 % activity when incubated in 50 °C for 1 h. Quantitative real-time PCR experiments demonstrated that SaFeSOD was expressed in leaf, stem, flower, fruit and root tissues with the highest expression in leaf tissues.     

Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: the mouse carnitine transporter OCTN3

pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni(2+)-chelating chromatography with a yield of 2mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [(3)H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K(m) for carnitine was 36 μM. [(3)H]carnitine transport was inhibited by carnitine analogues and some Cys and NH(2) reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.     

单纯疱疹病毒2gD-Hsp70融合蛋白基因的构建及表达

构建并原核表达Hsp70-HSV2gD融合蛋白。将Hsp70和HSV-2gD蛋白基因分别克隆到原核表达载体pGEX-4T-1,构建成重组质粒pGEX-4T-Hsp70-gD,并测序鉴定。重组质粒pGEX-4T-Hsp70-gD转化大肠杆菌DH5α后,IPTG诱导表达并进行SDS-PAGE分析。表达产物纯化后做Westernblot检测。将其肌注免疫BALB/c小鼠,检测融合蛋白对免疫小鼠脾淋巴细胞增殖、γ-干扰素产生以及血清中gDIgG水平的影响。表达产物的SDS-PAGE分析发现,在相对分子量为118kD处有外源蛋白表达,与预期蛋白带一致。用GST柱得到了纯化的Hsp70-HSV2gD融合蛋白。Westernblot证实,表达产物具有良好的活性。GST-Hsp70-gD组蛋白疫苗免疫的小鼠,其脾淋巴细胞刺激指数和脾淋巴细胞培养上清中γ-干扰素的水平高于其它组(P<0.05)。血清单纯疱疹病毒-2gD蛋白的抗体水平高于其它组(P<0.05)。     

Ⅰ群禽腺病毒33K基因的原核表达与鉴定

33K蛋白是Ⅰ群禽腺病毒(fowl adenovirus groupⅠ,FAVⅠ)的非结构蛋白,在感染的过程中起重要作用。为了构建原核表达载体pGEX-33K作为诊断抗原,本研究根据GenBank中FAVⅠ33K基因序列,设计1对特异性引物。用PCR方法扩增目的片段,将其克隆至原核表达载体pGEX-4T-1,构建了重组质粒pGEX-33K。将其转化大肠杆菌感受态细胞DH5α,经IPTG诱导和SDS-PAGE分析,pGEX-33K表达的分子量为36.7KD,表达产物主要以可溶性的形式存在于菌体中;最佳IPTG诱导浓度为0.75mmol/L,最佳诱导时间为4h;经Western Blot鉴定,pGEX-33K重组蛋白可与FAVⅠ阳性血清发生特异性反应,为FAVⅠ抗体ELISA鉴别诊断试剂盒的研究奠定基础。     

A hydrolytic ��-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

??-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with ??-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni²⁺-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t _1/2 of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Pt??-GT shared 37 % sequence identity and 53 % homology with an extremophile ??-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.     

GST-HRB融合蛋白的表达与纯化

构建GST-HRB重组质粒,进行融合蛋白的表达、纯化及鉴定.利用PCR扩增及基因重组技术,以pcDNA-3.1-HRB为模板扩增出HRB全基因序列,并将其插入带有GST(谷胱甘肽巯基转移酶)标签的原核表达载体pGEX-6P-1中,构建GST-HRB融合蛋白表达质粒.然后,将重组质粒GST-HRB转化至大肠杆菌Rosseta进行融合蛋白的表达.利用GST琼脂糖珠进行融合蛋白的纯化,最后应用SDS-PAGE电泳和Western blotting鉴定纯化的融合蛋白.结果表明,成功构建pGEX-6P-1-HRB原核表达载体,表达及纯化了GST-HRB融合蛋白.     

Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli

Antimicrobial peptides (AMPs) have been paid considerable attention owing to their broad-spectrum antimicrobial activity and have great potential as novel antimicrobials. In this study, a novel hybrid peptide LF15-CA8 was designed on the basis of bovine lactoferricin (LfcinB) and cecropin A. The gene segment encoding LF15-CA8 was synthesized and cloned into pGEX-4T-BH to form pGEX-4T-LC1 containing one copy of the LF15-CA8 coding region. A series of recombinant vectors containing up to six multiple-copy LF15-CA8 coding regions, i.e., pGEX-4T-LCn (n = 1–6), were subsequently constructed, and used for transformation in Escherichia coli BL21(DE3). After induction with IPTG, pGEX-4T-LC1 and pGEX-4T-LC2 transformants successfully expressed fusion proteins GST-LF15-CA8 and GST-(LF15-CA8)₂ in the form of inclusion bodies, respectively. The inclusion bodies were dissolved and the peptide was successfully released in 70 % formic acid in a single step. After purification, about 10.0 mg of the recombinant peptide LF15-CA8 with purity more than 97 % was obtained from 1 l of bacteria culture of pGEX-4T-LC2 transformants. LF15-CA8 caused an increase in antibacterial activity against Gram-positive bacterium (Staphylococcus aureus ATCC 25923) compared with the parent peptides and did not show obvious hemolytic activity against human erythrocytes in the range of effective antibacterial concentration. These results suggest that the peptide LF15-CA8 could be a promising candidate for therapeutic applications, and may lead to a cost-effective solution for the large-scale production of AMPs.     

人催乳素受体胞外区蛋白的原核表达及纯化

目的:建立人催乳素受体的原核表达系统,并在大肠杆菌中获得表达。方法:由RT-PCR获得人催乳素受体(human prolactin receptor,hPRLR)胞外区氨基酸的编码序列,扩增并通过酶切位点修饰后克隆至pMD18-T载体,经测序正确后,切下编码序列连接到重组表达载体pGEX-4T-2中,转化大肠杆菌Rosetta(DE3),用IPTG诱导重组工程菌表达,使用谷胱甘肽偶联的GSTrapFF柱亲和层析纯化重组蛋白。结果:重组菌株可以表达GST-hPRLR融合蛋白,用免疫印迹反应鉴定纯化的融合蛋白,在相对分子质量为37.6×103处有一条带。结论:利用大肠杆菌表达系统获得了较高纯度的GST-hPRLR融合蛋白,为进一步研究催乳素受体的功能和制备特异性的抗体奠定了基础。