Refolding and Characterization of a Yeast Dehydrodolichyl Diphosphate Synthase Overexpressed in Escherichia coli

Dehydrodolichyl diphosphate synthase (DDPPs) catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize long-chain dehydrodolichyl diphosphate, which serves as a precursor of glycosyl carrier in glycoprotein biosynthesis in eukaryotes. To perform kinetic and structural studies of DDPPs, we have expressed yeast DDPPs using Escherichia coli as the host cell. Thioredoxin and His tag were utilized to increase the solubility of the recombinant protein and facilitate its purification using Ni-nitrilotriacetic acid (NTA) column. The protein was overexpressed in E. coli but mostly existed in pellet in the absence of detergent. The low quantity of soluble DDPPs was purified using Ni-NTA, Mono Q anion-exchange, and size-column chromatographies. The protein in the pellet was solubilized with 7 M urea and purified using Ni-NTA under denaturing condition. The protein refolding was achieved via the stepwise dialysis to remove the denaturant in the presence of 6 mM β-mercaptoethanol. Detergent n-octyl-β- -glucopyranoside and Triton X-100 increased the solubility of the DDPPs so that refolding can be performed at higher protein concentration. Alternatively, on-column refolding was carried out in a single step to obtain the active protein in large quantities. β-Mercaptoethanol and Triton were both required in this quick refolding process. The kinetic studies indicated that the soluble and refolded DDPPs have comparable activities (k_cat = 2 × 10⁻⁴ s⁻¹). Unlike its bacterial homologue, undecaprenyl diphosphate synthase, yeast DDPPs activity was not enhanced by Triton.     

On-column purification and refolding of recombinant bovine prion protein: using its octarepeat sequences as a natural affinity tag

Prion protein has a key role in the occurrence of transmissible spongiform encephalopathy (TSE) and development of these diseases. Here, we provide a convenient procedure for on-column purification and refolding of the full-length mature bovine prion protein (bPrP) from Escherichia coli using immobilized metal (Ni) affinity chromatography, based on the metal-binding property of its unusual octarepeat sequences containing six tandem copies. Following extensive washing, the bPrP pellet was solubilized by guanidine hydrochloride and subjected to Ni-NTA agarose column. Purification and refolding were achieved by stepwise gradient washing with reduced guanidine hydrochloride concentrations. Triton X-100 and beta-mercaptoethanol were required in this rapid refolding process. The isolated prion protein was identified by monoclonal antibodies and its integrity was monitored by mass spectroscopy. Its correct folding was confirmed from circular dichroism (CD) experiments. Moreover, thioflavin T-binding assay showed that the recombinant bPrP could be transformed into amyloid fiber structures like that of the infectious prion isoform PrP(sc).     

Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens

A newly isolated gene from Agrobacterium tumefaciens (A. tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E. coli), and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da. The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources. The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases. High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography. E. coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E. coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A. tumefaciens dps gene is functionally expressed in E. coli and that it encodes a decaprenyl diphosphate synthase.     

Purification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli

The yeast ilv2 gene, encoding acetolactate synthase, was subcloned in an Escherichia coli expression vector. Although a major part of the acetolactate synthase synthesized by E. coli cells harbouring this vector was packaged into protein inclusion bodies, we used these recombinant E. coli cells to produce large quantities of the yeast enzyme. The yeast acetolactate synthase was purified to homogeneity using first streptomycin and ammonium sulfate precipitations, followed by T-gel thiophilic interaction, Sephacryl S-300 gel filtration, Mono Q anion exchange, and Superose 12 gel filtration chromatography. SDS/PAGE and gel filtration of the purified enzyme showed that it is a dimer composed of two subunits, each with the molecular mass of 75 kDa. The purified yeast acetolactate synthase was further characterized with respect to pH optimum, dependence of the substrate, pyruvate, and requirements of the cofactors, thiamin diphosphate, Mg2+, and FAD.     

Expression,oxidative refolding,and characterization of six-histidine-tagged recombinant human LECT2, a 16-kDa chemotactic protein with three disulfide bonds

Human LECT2 is a 16-kDa chemotactic protein that consists of 133 amino acids and three intramolecular disulfide bonds. Here, we present the oxidative refolding of (His)(6)-LECT2, an N-terminally (His)(6)-tagged recombinant protein of human LECT2. (His)(6)-LECT2 was overproduced in Escherichia coli in the form of insoluble aggregates, solubilized with 8 M urea in the presence of 10 mM DTT, and purified and refolded on Ni-NTA agarose by lowering the urea concentration before the elution. This process, however, gave a mixture of oligomers of (His)(6)-LECT2 as well as the monomer, whose composition was as low as 36%. The oligomers formed as a result of incorrect intermolecular disulfide bonds. After the refolding on Ni-NTA agarose (step 1), the disulfide bonds were shuffled using a glutathione redox buffer (step 2) and the remaining thiols were completely oxidized (step 3) to improve the yield of correctly folded, monomeric (His)(6)-LECT2. The monomer composition was significantly improved to 81% by the three-step refolding method and the monomer thus obtained was shown to have the same conformation as the authentic LECT2 produced in CHO cells by CD and NMR spectroscopies. The yield of (His)(6)-LECT2 was 1.0 mg/L E. coli culture and was 16 times as high as that in our previous report, in which (His)(6)-LECT2 was purified from the soluble fractions of E. coli cell lysates.     

重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。     

Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (相似文献   

何首乌中Ⅲ型聚酮合酶基因FmPKS2的原核表达及鉴定

目的：对传统中药何首乌中Ⅲ型聚酮合酶基因FmPKS2进行原核表达并鉴定重组蛋白酶活性,为研究该酶基因在何首乌有效成分代谢合成及其调控中的功能奠定基础。方法：根据何首乌FmPKS2（GenBank登录号：GQ984139）基因序列,通过PCR扩增其全长的编码区,克隆至原核表达载体PET-28a,构建重组质粒PET-FmPKS2,将其转化原核表达菌株E.coli BL21（DE3）,并用IPTG诱导表达,SDS-PAGE鉴定融合蛋白可溶性,通过Ni-NTA亲和树脂纯化可溶性蛋白后以丙二酰-COA和香豆酰-COA为底物进行催化反应,TLC鉴定反应产物。结果：经过诱导,重组菌表达分子量为42KD左右的融合蛋白,其中可溶性重组蛋白最优表达条件为IPTG浓度0.5 mmol/L,诱导时间6h,温度25℃。纯化后的可溶性重组蛋白催化丙二酰-COA和香豆酰-COA得到的产物经TLC鉴定为二苯乙烯类化合物白藜芦醇。结论：成功实现FmPKS2的原核表达且融合蛋白以可溶形式存在,催化反应证明FmPKS2融合蛋白具有二苯乙烯合酶的活性。     

Single-step purification and refolding of recombinant mouse and human myelin oligodendrocyte glycoprotein and induction of EAE in mice

The extracellular domain of human and rat MOG (ED-MOG) induces experimental autoimmune encephalomyelitis (EAE) when injected into susceptible animals. EAE is a T cell-mediated disease of the central nervous system commonly used as an animal model for human multiple sclerosis. Here, we describe a straightforward procedure for the purification and refolding of mouse and human ED-MOG overexpressed in Escherichia coli as inclusion bodies. Following solubilization and purification using Ni-NTA resin chromatography under denaturing conditions, a column-based refolding proceeded in renaturation buffer supplemented with a glutathione redox buffer system. Using this approach up to 33 mg of highly pure soluble proteins was obtained per liter of expression culture. The ability of purified proteins to induce EAE was evaluated in three strains of mice. We believe that the strategy described here would facilitate researchers to carry out encephalitogenic as well as structure-function studies of this autoantigen. Additionally, we show for the first time that mouse ED-MOG induces severe disease in mice.     

水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。     

金黄色葡萄球菌特异性PCR检测靶点的自动化筛选

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。     

Gene cloning and overexpression of a geranylgeranyl diphosphate synthase of an extremely thermophilic bacterium, Thermus thermophilus

A geranylgeranyl diphosphate (GGPP) synthase gene of an extremely thermophilic bacterium, Thermus thermophilus, was cloned and sequenced. T. thermophilus GGPP synthase, overexpressed in Escherichia coli cells as a glutathione S-transferase fusion protein, was purified and characterized. The fusion protein, retaining thermostability, formed a homodimer, and showed higher specific activity than did a partially purified thermostable enzyme previously reported. Optimal reaction conditions and kinetic parameters were also examined. The deduced amino acid sequence indicated that T. thermophilus GGPP synthase was excluded from the group of bacterial type GGPP synthases and lacked the insertion amino acid residues in the first aspartate-rich motif as do archaeal and eukaryotic short-chain prenyltransferases.     

Overproduction in Escherichia coli and purification of Epstein-Barr virus EBNA-1

Epstein-Barr virus nuclear antigen 1 (EBNA-1) is a multi-functional protein of the Epstein-Barr virus (EBV). Due to its low abundance in EBV-transformed cells, overproduction in a foreign host is preferred to obtain purified EBNA-1 protein. The EBNA-1 gene possesses a large number of Escherichia coli rare codons (23%). By using E. coli BL21(DE3)Rosetta2 cells that augment the low-abundance tRNA genes, the expression level of EBNA-1 in E. coli was greatly enhanced. EBNA-1 was then purified by applying the whole cell extract soluble fraction to a Ni-NTA Superflow column and eluting with an imidazole gradient. The improved overexpression in E. coli followed by a one-step Ni-NTA purification resulted in a sufficient amount of pure EBNA-1 protein to test DNA binding activity, and prepare and test EBNA-1-specific monoclonal antibodies (mAbs).     

Purification of insoluble nitric oxide synthase from rat cerebellum.

Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.     

Presence and removal of a contaminating NADH oxidation activity in recombinant maltose-binding protein fusion proteins expressed in Escherichia coli

We observed the presence of contaminating NADH oxidation activity in maltose binding protein (MBP) fusion proteins expressed in Escherichia coli and purified using conventional amylose resin-based affinity chromatography. This contaminating NADH oxidation activity was detectable with at least four different enzymes from Cryptosporidium parvum expressed as MBP-fusion proteins (i.e., an enoyl-reductase domain from a type I fatty acid synthase, a fatty acyl-CoA binding protein, the acyl-ligase domain from a polyketide synthase, and a putative thioesterase), regardless of their NADH dependence. However, contaminating NADH oxidation activity was not present when fusion proteins were engineered to contain a His-tag and were purified using a Ni-NTA resin-based protocol. Alternatively, for proteins containing only an MBP-tag, the contaminating activity could be eliminated through the addition of 0.1% Triton X-100 and 2% glycerol to the column buffer during homogenization of bacteria and first column wash, followed by an additional wash and elution with regular column and elution buffers. Removal of the artifactual activity is very valuable in the study of enzymes using NADH as a cofactor, particularly when the native activity is low or the recombinant proteins are inactive.     

Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity.

Comparison of the farnesyl diphosphate (FPP) synthase amino acid sequences from four species with amino acid sequences from the related enzymes hexaprenyl diphosphate synthase and geranylgeranyl diphosphate synthase show the presence of two aspartate rich highly conserved domains. The aspartate motif ((I, L, or V)XDDXXD) of the second of those domains has homology with at least 9 prenyl transfer enzymes that utilize an allylic prenyl diphosphate as one substrate. In order to investigate the role of this second aspartate-rich domain in rat FPP synthase, we mutated the first or third aspartate to glutamate, expressed the wild-type and mutant enzymes in Escherichia coli, and purified them to apparent homogeneity using a single chromatographic step. Approximately 12 mg of homogeneous protein was isolated from 120 mg of crude bacterial extract. The kinetic parameters of the purified wild-type recombinant FPP synthase containing the DDYLD motif were as follows: Vmax = 0.84 mumol/min/mg; GPP Km = 1.0 microM; isopentenyl diphosphate (IPP) Km = 2.7 microM. Substitution of glutamate for the first aspartate (EDYLD) decreased the Vmax by over 90-fold. The Km for IPP increased, whereas the Km for GPP remained the same in this D243E mutant. Substitution of glutamate for the third aspartate (DDYLE) did not result in altered enzyme kinetics in the D247E mutant. These results suggest that the first aspartate in the second domain is involved in the catalysis by FPP synthase.     

On-column refolding of an insoluble histidine tag recombinant exopolyphosphatase from Trypanosoma brucei overexpressed in Escherichia coli

An exopolyphosphatase gene has been cloned by polymerase chain reaction (PCR) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine tag) exopolyphosphatase in E. coli in order to characterize its biochemical activity and to produce antibody to determine its localization. Because overexpression of this protein in bacteria resulted in the formation of inactive inclusion bodies, these structures were first solubilized in denaturant condition (6 M urea). Secondly, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column from 6 M to 0 M urea in the presence of 1% Triton X-100. Triton X-100 was used to abolish protein aggregation during the refolding step. The purified enzyme was active, demonstrating that at least part of the proteins was properly refolded.     

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.     

Expression and Purification of the DNA Binding Domain of the Epstein-Barr Virus Nuclear Antigen 1

To overexpress EBNA-1 in E.coli and generate the specific antibody,in this study,the antigenicity,epitope and hydrolysis of EBNA-1 were analyzed using the computer design software Biosun.Based on the prediction by computer analysis,the sequence encoding EBNA-1385-557 was amplified by PCR with the specific primers.The expression vector containing EBNA-1385-557 coding sequence was constructed.His-tagged EBNA-1385-557 was expressed in E.coli.The soluble recombinant protein was purified using Ni-NTA chromato...