一个高效表达、快速纯化大肠杆菌精氨酰-tRNA合成酶的新系统(英文)

编码大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶（ Ａｒｇ Ｒ Ｓ） 的基因ａｒｇ Ｓ 被克隆到ｐ Ｍ Ｆ Ｔ７５ 载体上。将此质粒转化的大肠杆菌 Ｊ Ｍ１０９（ Ｄ Ｅ３） 中, 该转化子粗抽液的比活是宿主菌的２ ５００ 倍。通过 Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ Ｆａｓｔ Ｆｌｏｗ 和 Ｂｌｕｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ两步柱层析在一天内即可将精氨酰ｔ Ｒ Ｎ Ａ 合成酶纯化至电泳一条带, 比活为３６ ０００ ｕ／ｍｇ , 总收率可达６９ ％ 。与以前报道的 Ａｒｇ Ｒ Ｓ的高表达质粒相比, 使用该重组质粒可以很方便地将昂贵的标记氨基酸高效地参入酶分子内。目前的研究结果表明,该新系统能够很方便地提供大量的更高比活的大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶以进行该酶的 Ｎ Ｍ Ｒ 和结晶学研究     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.

Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.     

一种插入116个氨基酸的亮氨酰—tRNA合成酶具有高酶活力

大肠杆菌亮氨酰 tRNA合成酶 (LeuRS)是第 1类氨基酰 tRNA合成酶 ,由 860个氨基酸残基组成 ,催化亮氨酸tRNA的亮氨酰化。研究发现 ,在它的CP1结构域内 3 68和 3 69间的肽键间插入 2 5 3～ 3 68的肽段 ,该插入变种的酶仍具有酶活力 ,取名为LeuRS C。由于这一插入变种的不稳定性 ,构建了His6 LeuRS C的表达质粒 ,用Ni NTA柱亲和层析的方法进行纯化。发现His6 LeuRS C虽然插入了 116个氨基酸残基 ,但仍具有全部的天然LeuRS的活力。测定了His6 LeuRS C的酶学动力学常数 ,比较了它与天然LeuRS的从CD光谱得到的二级结构和热稳定性     

Molecular cloning and nucleotide sequence of the gene for Escherichia coli leucyl-tRNA synthetase.

The gene for Escherichia coli leucyl-tRNA synthetase leuS has been cloned by complementation of a leuS temperature sensitive mutant KL231 with an E.coli gene bank DNA. The resulting clones overexpress leucyl-tRNA synthetase (LeuRS) by a factor greater than 50. The DNA sequence of the complete coding regions was determined. The derived N-terminal protein sequence of LeuRS was confirmed by independent protein sequencing of the first 8 aminoacids. Sequence comparison of the LeuRS sequence with all aminoacyl-tRNA synthetase sequences available reveal a significant homology with the valyl-, isoleucyl- and methionyl-enzyme indicating that the genes of these enzymes could have derived from a common ancestor. Sequence comparison with the gene product of the yeast nuclear NAM2-1 suppressor allele curing mitochondrial RNA maturation deficiency reveals about 30% homology.     

Human mitochondrial leucyl-tRNA synthetase with high activity produced from Escherichia coli

The processing of human mitochondrial leucyl-tRNA synthetase had been previously investigated in insect cell. In the present work, the gene encoding human mitochondrial leucyl-tRNA synthetase with the same N-terminus as that processed in the mitochondria of insect cell was cloned and expressed in Escherichia coli. The enzyme was purified by affinity chromatography on Ni-NTA column. About 6 mg of human mitochondrial leucyl-tRNA synthetase was obtained from 1 liter of culture. The specific activity of the purified enzyme is 127.7 units/mg, the highest activity of the reported results; this enzyme has the potential for characterizing the mitochondrial tRNA mutants associated with some human mitochondrion-related neuromuscular disorders. The kinetic constants for three substrates: leucine, ATP, and E. coli tRNA1Leu (CAG) in the leucylation reaction are also reported herein.     

Expression and simple,one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in <Emphasis Type="Italic">Escherichia coli</Emphasis> and their interaction with protoporphyrin IX

The product of human fragile histidine triad (FHIT) gene is a tumor suppressor protein of still largely unknown cellular background. We have shown previously that it binds protoporphyrin IX (a photosensitizer) which alters its enzymatic activity in vitro. Fhit, diadenosine triphosphate (Ap₃A) hydrolase, possesses the active site with histidine triad His-φ-His-φ-His-φφ. So-called histidine Fhit mutants (His94Asn, His96Asn and His98Asn) exhibit highly reduced activity in vitro, however, their antitumor function has not been fully described yet. In this work we have cloned the cDNAs of histidine mutants into pPROEX-1 vector allowing the production of His₆-fusion proteins. The mutated proteins: Fhit-H94N, Fhit-H96N and Fhit-H98N, were expressed in Escherichia coli BL21(DE3) and purified (up to 95%) by an improved, one-step affinity chromatography on Ni-nitrilotriacetate resin. The final yield was 2 mg homogenous proteins from 1 g bacteria (wet wt). The activity of purified proteins was assessed by previously described assay. The same purification procedure yielded 0.8 mg/ml and highly active wild-type Fhit protein (K _m value for Ap₃A of 5.7 μM). Importantly, purified mutant forms of Fhit also interact with a photosensitizer, protoporphyrin IX in vitro.     

丙酮酸甲酸裂解酶基因在大肠杆菌中的克隆、表达及酶学性质的研究

丙酮酸甲酸裂解酶（pyruvate format-lyas,PFL）是厌养或兼性厌养微生物中,代谢途径的关键酶之一,为了进一步研究其功能,我们以大肠杆菌JM109菌株基因组DNA为模板,进行PCR扩增大肠杆菌中的pfl基因,为测序方便将所得DNA片段连接到pMD18-T载体上,将测序正确后的pfl基因连接到表达载体pET-22b(＋)中,重组表达载体在大肠杆菌BL21(DE3)中诱导表达, 通过SDS-PAGE电泳分析,在分子量为85kDa处出现新生的蛋白条带。利用金属亲和层析对添加了6×组氨酸标签的PFL进行纯化,对PFL的酶学性质进行了研究。结果表明：此酶的最适温度为35 ℃,最适pH为7.5,米氏常数Km＝2.3mmol,Tm＝49.9℃。     

Isolation and Partial Characterization of Temperature-Sensitive Escherichia coli Mutants with Altered Leucyl- and Seryl-Transfer Ribonucleic Acid Synthetases

Two temperature-sensitive mutants of Escherichia coli have been found in which the conditional growth is a result of a thermosensitive leucyl-transfer ribonucleic acid (tRNA) synthetase and seryl-tRNA synthetase, respectively. The corresponding genetic loci, leuS and serS, cotransduce with lip and serC, respectively. As a result of the mutationally altered leucyl-tRNA synthetase, some leucine-, valine-, and isoleucine-forming enzymes were derepressed. Thus, leucyl-tRNA synthetase is involved in the repression of the enzymes needed for the synthesis of branched-chain amino acids.     

Gluconobacter oxydans NH-10中短链D-阿拉伯糖醇脱氢酶的研究

以氧化葡萄糖酸杆菌(Gluconobacter oxydans)NH-10基因组DNA为模板,扩增得到D-阿拉伯糖醇脱氢酶基因arDH,将其克隆到大肠杆菌表达载体JM109(DE3)中进行诱导表达。SDS-PAGE电泳分析ArDH的分子量约为30 kDa,是一个短链脱氢酶,既能催化D-阿拉伯糖醇氧化为D-木酮糖,又能催化D-木酮糖还原为D-阿拉伯糖醇。催化氧化反应时,对D-阿拉伯糖醇的K_m为60.67 mmol/L,V_max为0.803 U/mg;它能同时依赖于NAD⁺和NADP⁺,但是更加偏好辅酶NAD⁺;最适pH为12.0。还原反应对D-木酮糖的 K_m为36.39 mmol/L,V_max为1.71 U/mg;最优pH为7.0,最适温度均为30℃。     

Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.     

arcA基因提高大肠杆菌对有机溶剂的耐受性

【目的】将来源于恶臭假单胞菌(Pseudomonas putida JUCT1)的基因arc A(编码精氨酸脱亚胺酶)整合到Escherichia coli JM109(DE3)基因组中,以提高该菌对有机溶剂的耐受性。【方法】以P.putida JUCT1的基因组为模板扩增基因arc A,并与p ET-20b(+)连接后导入E.coli JM109(DE3)中,验证该基因提高E.coli JM109(DE3)对有机溶剂的耐受性。利用Red同源重组的方法将arc A整合到E.coli JM109(DE3)基因组中。【结果】E.coli JM109(DE3)/p ET-20b(+)-arc A在添加了2.0%(体积比)环己烷、0.1%(体积比)甲苯、4.0%(体积比)萘烷和0.1%(体积比)丁醇的培养基中培养8 h后,其OD660由初始的0.2分别上升到0.8、0.9、1.8和1.3。将arc A成功整合到E.coli JM109(DE3)基因组中,获得了具有较好遗传稳定性的溶剂耐受E.coli JM109(DE3)宿主菌株。【结论】外源基因arc A能提高大肠杆菌菌株的有机溶剂耐受性,为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。     

利用温控载体构建碱性果胶酯裂解酶工程菌

从筛选出的Bacillus subtilisWSHB04-02菌株中扩增出编码碱性果胶酯裂解酶的结构基因PL,将其插入载体pET22b( )多克隆位点,得到带有前导序列PelB重组质粒pET22b( )PL。以pET22b( )PL为模板扩增出带前导序列的碱性果胶酯裂解酶的结构基因PL,将其插入温控载体pHsh,重组载体在大肠杆菌JM109中得到表达。其表达量与以T7为启动子的重组菌BL21DE3[(pET22b( )PL]相比,表达量相近。SDS-PAGE分析显示表达产物的分子量均为43kDa,同核酸序列测定所推导的值相符。研究表明利用pHsh构建的JM109(pHsh PL)诱导表达好,诱导方式简单廉价,这对该酶的大规模发酵具有重要意义。     

Production of l-phenylalanine from trans-cinnamic acids by high-level expression of phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing P_LP_R promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.     

Expression,purification, and characterization of human tyrosyl-tRNA synthetase

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.     

Expression, purification, and characterization of His-tagged Staphylococcus xylosus lipase wild-type and its mutant Asp 290 Ala

The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.     

改造稀有密码子提高SEA蛋白表达量

利用重叠PCR技术突变了sea基因上一个稀有密码子簇,将此段中稀有密码子全部更换成E.coli最常用密码子,得到sea^m。将sea和sea^m分别克隆于7ZTS表达载体上,并转化JM109（DE3）菌株。结果表明,sea基因的表达十分微弱,而sea^m基因的表达量十分高,约占菌体总蛋白的15 ％。表达产物在体内具有一定的抗肿瘤活性。     

Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression.

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.     

phbB,phbC Gene Expression in E. coli and Their Transformation and Identification in Potato

Using NADPH-dependent acetoacetyl-CoA reductase gene (phbB) and poly-β-hydroxybutyrate (PHB) synthase gene (phbC) cloned from Alcaligenes eutrophus H16 and expression vector pKK223-3, the authors constructed an E. coli expression vector pKCB containing independent phbB and phbC operators, respectively, and transfered it into E. coli JM109. The microscopy and GC analysis indicated that E. coli JM109 (containing pKCB) induced by IPTG could synthesize poly-β- hydroxybutyrate (PHB). By DNA processing, three tuber-specific plant expression vectors, pP- SAGB (containing phbB), pBIBGC ( containing phbC) and pPSAGCB ( containing both phbB and phbC), were successfully constructed. In 5 transformed potato cuhivars, the authors screened 20 positive lines.