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1.
Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 34 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1-103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO(-)(3) and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc. 相似文献
2.
Yeast protein Yol066 (encoded by YOL066 ORF, also known as Rib2) possesses two distinct sequence domains: C-terminal deaminase domain and N-terminal part related to RNA:pseudouridine (psi)-synthases. The deaminase domain is implicated in the riboflavine biosynthesis, while the exact function of the RNA:Psi-synthase domain remains obscure. Here we report the optimisation of growth conditions and purification scheme for recombinant His(6)-tagged Yol066 expressed in E. coli BL21(DE3) using pET28 plasmid. Production of soluble Yol066 protein is best at low temperature (18 degrees C) and IPTG concentration (50 micro M) and Yol066 purification was achieved using metal-affinity and ion-exchange chromatography. This optimised protocol yields about 10 mg of highly purified recombinant Yol066 from 3 l of E. coli culture. 相似文献
3.
通过反转录PCR获得了SARS冠状病毒核衣壳蛋白(N)和膜蛋白(M)基因,其序列分析结果与加拿大多伦多株完全一致。将M基因和N基因克隆到大肠杆菌表达载体pET22b和pBV222上,并在大肠杆菌中以包涵体及可溶形式获得高效表达。通过离子交换、金属螯合层析纯化获得电泳纯制品。所获得的核衣壳蛋白具有良好的抗原性,可用于抗SARS抗体检测及亚单位疫苗研究。 相似文献
4.
目的:通过在大肠杆菌SUMO系统中对鼠双微体2(MDM2)C端结构域ZFRING(aa.300-491)进行构建并进行表达,酶切和纯化,从而得到MDM2蛋白C端结构域的单体结构,为其后续的晶体研究及MDM2非p53依赖途径的研究提供途径。方法:利用大肠杆菌SUMO表达系统对zfring基因进行重组构建。构建成功的表达载体经诱导表达优化后,通过Ni-NTA进行亲和层析纯化,并利用SDS-PAGE及Western blot鉴定分析。纯化后的融合蛋白经ULP1酶切得到目的蛋白ZFRING,并通过Hi Trap Q FF离子交换层析检验和去除杂质DNA。最后通过分子筛检验其蛋白结构。结果:构建了SUMO-ZFRING重组载体。重组载体在大肠杆菌高效可溶性表达,纯化并酶切后的目的蛋白ZFRING以单体形式存在。结论:通过原核表达、纯化、酶切及层析发鉴定,成功获得高稳定、高纯度且为单体结构的MDM2 C端结构域ZFRING蛋白,为后续关于MDM2,尤其是其非p53依赖途径的结构学和功能学提供了思路和途径。 相似文献
5.
筛选一种高效重组金黄色葡萄球菌蛋白A(SpA)用于制备抗体纯化亲和介质。首先通过基因操作获得金黄色葡萄球菌蛋白A(SpA)的Z结构域单体、二串体、三串体、四串体和五串体基因,将目的基因分别克隆至pET-22b表达载体并转化至大肠杆菌BL21(DE3)感受态细胞,获得不同串联个数的Z结构域基因工程菌,经诱导表达和Ni2+亲和层析纯化得到Z结构域单体和二-五串体蛋白。纯化后的目的蛋白偶联至琼脂糖凝胶作为亲和层析介质,对人免疫球蛋白G(IgG)进行分离纯化。分析比较Z结构域串联体蛋白产量及其偶联的亲和介质对抗体吸附载量的差异。结果表明,构建的Z结构域单体、二串体、三串体、四串体和五串体基因工程菌能有效表达目的蛋白,制备的凝胶亲和介质可特异性吸附人IgG。增加Z结构域串联数,重组蛋白产量和单位摩尔数多聚体蛋白吸附载量获得提高,其中,重组四串体蛋白产量大(160 mg/10 g湿菌体),对抗体的吸附载量高(34.4 mg人IgG/mL胶),更适合作为配基用于亲和层析介质的制备。 相似文献
6.
Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed. 相似文献
7.
Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal
region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1–103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO −3 and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc. 相似文献
8.
Escherichia coli alpha-hemolysin (AH) purified from culture supernatants by gel filtration and ion-exchange chromatography was heterogeneous in charge and size. A 107,000-dalton protein was identified as the product of the hlyA gene by its reactivity with anti-AH monoclonal antibodies. Proteolysis of the product of the hlyA gene occurred but was not required for transport of the protein through the cell wall. Active AH had a larger size and lower pI than analysis of the hlyA gene sequence would predict, thus suggesting that the hlyA protein is complexed with other bacterial products. Lipopolysaccharide was detected in purified hemolysin complex preparations and may be a major component of the complexes. These findings suggest several possible mechanisms for release of AH from the bacterial cell including release by outer membrane fragmentation. The existence of AH complexed with lipopolysaccharide may have important implications in understanding its toxicity. 相似文献
9.
Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence. 相似文献
10.
ShlB from Serratia marcescens was isolated and purified from a porin-deficient Escherichia coli BL21 strain using a combination of detergent extraction, affinity and ion-exchange chromatography. An internal histidine affinity tag was introduced that did not interfere with activity. At each stage of the purification scheme biological activity of the ShlB protein was assessed. Using this scheme, several His(6)-tagged mutants of ShlB were purified to electrophoretic homogeneity. 相似文献
11.
目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。 相似文献
12.
A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method. 相似文献
13.
Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues. 相似文献
14.
血管生长抑制因子Kringle 5 是目前发现的抑制血管内皮细胞增殖和肿瘤生长的活性最强的纤溶酶原片段,特异性高而毒副作用小,在肿瘤的治疗方面具有潜在的巨大价值和广阔的应用前景。根据K5基因的序列设计PCR引物,通过PCR从已有的克隆载体扩增出人纤维蛋白溶酶原的K5部分基因,将K5基因克隆入原核表达载体pET15b,经序列测定,成功构建了pET15b-K5非融合表达载体。将重组载体导入大肠杆菌中IPTG诱导表达,SDS-PAGE分析目的蛋白主要以可溶形式存在于菌体中,破碎后上清通过阳离子交换层析,纯化获得纯度大于95%的目标蛋白,脱盐后对分子量测定推测形成了三聚体。通过鸡胚绒毛尿囊膜法证明蛋白产物对鸡胚绒毛尿囊膜血管增生有一定的抑制作用。 相似文献
15.
In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme. 相似文献
16.
Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC50 was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli. 相似文献
17.
We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85 degrees C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA. 相似文献
18.
Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity. After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts. Recombinant human bFGF 1-146 and [Ala69,Ser87]bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor. Substitution of aspartic acid-19 by arginine in the first heparin-binding domain yielded a molecule that stimulated a higher total mitogenic response in fibroblasts as compared to bFGF. In addition, replacement of either arginine-107 in the second domain or glutamine-123 in the third domain with glutamic acid resulted in compounds that were 2 and 4 times more potent than bFGF. In contrast, substitution of arginine-107 with isoleucine reduced the activity of the molecule by 100-fold. Combination of domain substitutions to generate the [Glu107,123]bFGF and [Arg19,Lys123,126]bFGF mutants did not show any additivity of the mutations on biological activity. Alterations in the biological activity of the analogues was dependent on both the site of and the type of modification. Increased positive charge in the first domain and increased negative charge in the second and third domains enhanced biological potency. The altered activities of the derivatives appear to be due in part to changes in the affinity of the analogues for heparin. We conclude that changes in all three of the putative heparin-binding domains result in altered mitogenic activity and heparin interaction of basic fibroblast growth factor. 相似文献
19.
A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria. 相似文献
20.
Campylobacter is now recognized as the most common bacterial agent of gastroenteritis. The adhesion of bacteria to intestinal cells is a major step in human colonization. The binding of Campylobacter jejuni cells to fibronectin (Fn), a component of the extra cellular matrix, is mediated by a 37,000 outer membrane protein termed CadF for Campylobacter adhesion to Fn. CadF protein is very hard to purify from Campylobacter membranes. In order to study the conformation of this protein, we set out to clone, express, purify, and re-fold the CadF protein. The nucleotide sequence encoding the N-terminal domain of the CadF protein was cloned in a pET-based expression vector. The recombinant protein was further produced in Escherichia coli, purified from inclusion bodies, and refolded. More specifically, the purification experiments were set-up as follows: (i) protein aggregates were collected from cell-lysates, solubilized in urea and enriched by ion-exchange chromatography; (ii) refolding was achieved by drop-by-drop dilution method in detergent containing buffer and monitored by CD measurements; (iii) the protein was finally purified to homogeneity by gel filtration chromatography. In spite of our success in purifying the N-terminal domain of the CadF protein, repeated attempts to express and purify the entire cadF gene in E. coli failed. Using a novel approach, we found it possible to express the entire cadF gene fused to a hexa-histidine encoding nucleotide sequence in C. jejuni. This allowed the expression, synthesis, and purification of the recombinant CadF-His tagged protein from C. jejuni by nickel affinity chromatography followed by gel filtration chromatography. In summary, we developed a novel strategy to produce significant quantities of a recombinant N-terminal portion of the CadF protein (46.5 microg/mg of bacterial dry weight) and of the native CadF protein (3.5 microg/mg of bacterial dry weight) for further studies. 相似文献
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