片球菌素pediocin PA-1基因在大肠埃希菌中的融合表达

目的构建编码细菌素pediocin PA-1基因片段的原核表达载体,诱导表达,鉴定表达产物。方法重组DNA技术构建原核表达载体pET32-papA,IPTG诱导表达,用金属亲和层析纯化,并通过SDS-PAGE与Westernblot对表达的重组蛋白进行鉴定。结果双酶切和测序鉴定显示papA片段插入正确,诱导表达后获得分子量为19 kD的融合蛋白,表达量为25%,用金属亲和层析的方法获得纯化的片球菌素pediocin PA-1。结论papA基因片段编码的蛋白质能在原核细胞中正确表达,为下一步研究该功能域的生物活性奠定了基础。     

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.     

Chicken deoxyribonuclease: purification,characterization, gene cloning and gene expression

Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn²⁺ and Mg²⁺ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu²⁺, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca²⁺ at pH 8, chicken DNase resisted to the trypsin or -mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5-untranslated region and 166 of the 3 and, in the 5-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 ± 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates.     

Soluble expression, purification, and characterization of Gloydius shedaoensis venom gloshedobin in Escherichia coli by using fusion partners

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, is usually produced as inclusion bodies in Escherichia coli cell. In this work, gloshedobin was separately fused with three fusion partners NusA, GST, and TrxA at its N terminus and then was expressed as fusion proteins in E. coli. The results showed that the NusA was the most efficient fusion partner to improve the solubility of recombinant gloshedobin. The purified NusA-fused gloshedobin with an overall yield of 64.6% was resolved as one band in the SDS-PAGE gel with molecular mass of about 90 kDa. Both fibrinogen clotting and fibrinogenolytic activities were found for the recombinant product. The purified NusA-fused gloshedobin exhibited amidolytic activity of 506 U/mg under optimal conditions of pH of 8.0 and 40°C. The inhibition study of NusA-fused gloshedobin by various inhibitors showed that serine protease inhibitors, phenylmethylsulphonyl fluoride, and N-tosyl-l-phenylalanine chloromethyl ketone, strongly inhibited its admidolytic activity, whereas ethylenediaminetetraacetic acid as well as heparin and hirudin did not, suggesting that NusA-fused gloshedobin exhibited the same characteristics as the native form of gloshedobin. The strategy of this work may contribute to improve the soluble expression level of other thrombin-like enzymes from snake venom in E. coli.     

Synthesis of 32P-labelled protein probes using a modified thioredoxin fusion protein expression system in Escherichia coli

The thioredoxin fusion protein expression system from invitrogen was modified so that 32P-labelled recombinant proteins can be easily obtained in large quantities for functional studies. Proteins that are prone to form the inclusion bodies can be functionally expressed as thioredoxin fusion proteins in Escherichia coli. After expression, the recombinant proteins can be easily phosphorylated with 32P-gamma ATP and the 32P-labelled protein can be obtained functionally via a mild proteolytic digestion to cleave off the thioredoxin moiety. A deletion construct of the Ah receptor nuclear translocator protein was used as an example to illustrate how this protein expression system works.     

Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression.

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome.     

Gene cloning,purification, and characterization of 2,3-diaminopropionate ammonia-lyase from Escherichia coli

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes alpha,beta-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5'-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the alpha,beta-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D-and L-beta-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.     

Thermostable D-carbamoylase from Sinorhizobium morelens S-5: purification, characterization and gene expression in Escherichia coli

A d-carbamoylase from Sinorhizobium morelens S-5 was purified and characterized. The enzyme was purified 189-fold to homogeneity with a yield of 19.1% by aqueous two-phase extraction and two steps of column chromatography. The enzyme is a homotetramer with a native molecular mass of 150 kDa and a subunit relative molecular mass of 38 kDa. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. The enzyme showed high thermal and oxidative stability. It was found to have a K(m) of 3.76 mM and a V(max) of 383 U/mg for N-carbamoyl-d-p-hydroxyphenylglycine. The hyuC gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of d-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity from the recombinant. Our results show that the enzyme has great potential for industrial application.     

一种新的DyP-type过氧化物酶在大肠杆菌中的重组表达、纯化及鉴定

染料脱色过氧化物酶(DyP-type过氧化物酶)是含有亚铁血红素,能降解各种有毒染料的一类蛋白.为了研究运动发酵单胞菌Zymomonas mobilis ZM4 (ATCC 31821)中一种新的DyP-type过氧化物酶的特点和功能,以Z.mobilis基因组DNA为模板,通过PCR扩增目的基因,克隆到大肠杆菌表达载体pET-21b(+)中.通过ZmDyP与其他DyP-type过氧化物酶的比对,发现它们存在着共同保守氨基酸D149、R239、T254、F256和GXXDG结构基序,说明ZmDyP是Dyp-type过氧化物酶家族的一个新成员.经IPTG诱导大肠杆菌中pET21 b(+)-ZmDyP表达,并将表达的酶进行金属螯合层析纯化.SDS-PAGE分析表明,纯酶分子量为36 kDa,而活性染色显示分子量为108 kDa,表明该酶在活性状态下可能是一个三聚体.光谱扫描显示ZmDyP有一个典型的亚铁血红素吸收峰,说明它是含有亚铁血红素的蛋白.对ZmDyP性质进行了研究,发现以2,2-二氨-双(3-乙基苯并噻唑-6-磺酸)ABTS为底物,ZmDyP表现出更高的转化效率.这些研究结果丰富了DyP-type 过氧化物酶家族信息,并且为ZmDyP的结构功能和反应机制研究奠定了基础.     

A beta-mannanase from Bacillus subtilis B36: purification, properties, sequencing, gene cloning and expression in Escherichia coli

MANB36, a secrete endo-beta-1,4-D-mannanase produced by Bacillus subtilis B36, was purified to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 degrees C. The enzyme activity of MANB36 is remarkably thermostable at 60 degrees C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoethanol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted molecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.     

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.     

cDNA cloning, expression, purification, distribution, and characterization of biologically active canine alanine aminotransferase-1

Alanine aminotransferase (ALT) is a pyridoxal enzyme found mainly in the liver and kidney, but also in small amounts in the heart, muscle, fat, and brain. Serum aminotransferase activities have been used broadly as surrogate markers for tissue injury and disease in human and veterinary clinical settings and in safety assessment of chemicals and pharmaceuticals. Because of its relative abundance in liver, increased serum ALT activity is generally considered indicative of liver damage. Two ALT isoenzymes, ALT1 and ALT2, are known and have been cloned and sequenced from human, rat, and mouse. In this study, we have cloned the complementary DNA encoding the canine orthologue of ALT1 (cALT1). The complete cDNA sequence comprised 1852 bases and contained a 1485-base open reading frame, which encodes a polypeptide of 494 amino acid residues. Canine ALT1 shares 87.7, 87.2, and 87.0% amino acid identity to its human, mouse, and rat orthologues, respectively. The cDNA was expressed in Escherichia coli, with a N-terminal His (6x) tag, and the recombinant enzyme was purified using immobilized metal-affinity chromatography. The final yield of the purified recombinant cALT1 was greater than 5mg/L culture. The alanine transaminase activity of purified cALT1 was 229.81U/mg protein, which is approximately 38-fold higher than that of total soluble recombinant E. coli cell lysate, confirming that the enzyme is a functional ALT. Evaluation of various canine tissues by RT-PCR revealed that the level of ALT1 expression is in the order of: heart>liver>fat approximately brain approximately gastrocnemius>kidney. The purified cALT1 will be helpful to develop isoenzyme-specific anti-bodies, which could further improve the diagnostic resolution of current ALT assays in drug safety studies.     

Production of biologically active thymulin in Escherichia coli through expression of a chemically synthesized gene

Summary A synthetic gene coding for thymulin was ligated into an expression vector (pJB 1301) and placed under lac operon control. In the recombinant clones, thymulin was expressed as part of a galactosidase chimeric protein which was then cleaved by cyanogen bromide. Thymulin was purified using various chromatography systems including gel filtration and HPLC, and was detected by radioimmunoassay (RIA). In the biological assay the purified recombinant peptide demonstrated the same zinc dependency as natural thymulin and had the same amino acid composition and primary structure.     

Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.     

黑曲霉硫氧还蛋白的克隆表达纯化及其酶活位点特征分析

首次从黑曲霉Aspergillus niger全基因组中克隆出黑曲霉硫氧还原蛋白基因AnTrx,并对其编码蛋白的第33-37位保守区的活性位点实施定点突变C34S、C37S及C34S-C37S,获得相应的3个定点突变基因。将野生型AnTrx及其突变子分别在大肠杆菌Escherichia coli中诱导表达,比浊法测定纯化的各表达产物还原牛胰岛素α与β链之间二硫键的活性。结果表明,AnTrx的3个突变体都不表现明显催化活性。当突变型与野生型AnTrx等量混合后,发现突变型AnTrx-C34S可显著提高野生型AnTrx的催化效率,而突变型AnTrx-C37S却无此功能。由此证明,AnTrx活性结构域的第37位Cys残基上的巯基能参与攻击硫氧还蛋白和底物蛋白所形成的二硫键而释放被还原的底物蛋白,而第34位Cys残基同其他微生物的同一活性域一样参与硫氧化还蛋白与底物的结合。这一结果有助于认识真菌硫氧还蛋白第37位活性位点的作用。     

The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.     

Expression,purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli

Enterokinase (EC 3.4.21.9) is a serine proteinase in the duodenum that exhibits specificity for the sequence (Asp)(4)-Lys. It converts trypsinogen to trypsin. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for in vitro cleavage of fusion proteins. cDNA encoding the catalytic chain of Chinese bovine enterokinase was cloned and its encoding amino acid sequence is identical to the previously reported sequence although there are two one-base mutations which do not change the encoded amino acid. The EK catalytic subunit cDNA was cloned into plasmid pET32a, and fused downstream to the fusion partner thioredoxin (Trx) and the following DDDDK enterokinase recognition sequence. The recombinant bovine enterokinase catalytic subunit was expressed in Escherichia coli BL21(DE3), and most products existed in soluble form. After an in vivo autocatalytic cleavage of the recombinant Trx-EK catalytic domain fusion protein, intact, biologically active EK catalytic subunit was released from the fusion protein. The recombinant intact EK catalytic subunit was purified to homogeneity with a specific activity of 720 AUs/mg protein through ammonium sulfate precipitation, DEAE chromatography, and gel filtration. The purified intact EK catalytic subunit has a K(m) of 0.17 mM, and K(cat) is 20.8s(-1). From 100 ml flask culture, 4.3 mg pure active EK catalytic subunits were obtained.     

Molecular cloning,characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.