Overexpression, Purification, and Refolding of a Porphyromonas gingivalis Cysteine Protease from Escherichia coli

This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease.     

黑曲霉脂肪酶：基因克隆、表达及体外复性

根据黑曲霉F044脂肪酶N-端氨基酸序列,运用生物信息学方法,找到与黑曲霉脂肪酶基因同源的候选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044bp,含3个内含子,编码297个氨基酸(含信号肽27个氨基酸),与其它脂肪酶基因没有明显同源性。将编码成熟脂肪酶的anl连接到pET28a载体上得到重组表达质粒,转化大肠杆菌BL21(De3),诱导表达并纯化出目的蛋白。通过大量稀释和DEAESepharoseFastFlow层析相结合的方法,变性后的纯化蛋白在体外实现再折叠复性。     

Effective Purification of Nonspecific Cross-Reacting Antigens with Phosphatidylinositol-Specific Phospholipase C

ABSTRACT

Two molecular species of nonspecific cross-reacting antigens, NCA-90 and NCA-50 with mol. wts. of 90,000 and 50,000, respectively, wereeffectively extracted with phosphatidylinositol-specific phospholipase C (PI-PLC) from human lung tissues, followed by extraction with perchloric acid, immunoaffinity chromatography with anti-NCA adsorbent, and gel filtration on a TSK G3000SW column. The yields of NCA were about 2 times more than those obtained by the usual method without PI-PLC. Addition of 0.05 unit of PI-PLC to 1 g of lung tissue and incubation at 37°C for 1 h with continuous shaking seem to be practically sufficient for NCA extraction. The immunochemical properties of the NCAs thus obtained were found to be identical to those of NCAs obtained by the ordinary method.     

Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis

Summary A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec⁺ and Rec^- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U¹⁴C] sucrose and the growth of the cells.It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.     

Purification, Characterization and Cloning of Phospholipase D from Peanut Seeds

We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K _m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl₂ and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.     

拟南芥非特异性磷脂酶C4基因表达模式的研究

拟南芥非特异性磷脂酶C4（AtNPC4）具有降解磷脂酰胆碱（PC）,产生二酰甘油（DAG）和磷酸胆碱的活性。本研究从拟南芥基因组中分离了NPC4基因起始密码子上游1 379bp的启动子序列,与GUS报告基因融合后转化拟南芥,获得转基因植株。GUS组织化学染色表明,AtNPC4基因主要在处于衰老过程中的叶片中高水平表达,在根、茎、种荚和花中也有一定程度的表达,这种表达模式与RT-PCR结果相一致。另外,通过RT-PCR发现,AtNPC4基因在转录水平上受脱落酸的诱导,但不受水杨酸和茉莉素诱导。     

Overexpression and Purification of CytochromecOxidase fromRhodobacter sphaeroides

Theaa₃-type cytochromecoxidase ofRhodobacter sphaeroideshas been overexpressed up to seven fold over that in wild-type strains by engineering a multicopy plasmid with all the required oxidase genes and by establishing optimum growth conditions. The two operons containing the three structural genes and two assembly genes for cytochromecoxidase were ligated into a pUC19 vector and reintroduced into several oxidase-deletedR. sphaeroidesstrains. Under conditions of relatively high pH and maximal aeration, high levels of expression were observed. A smaller expression vector, pBBR1MCS, and a fructose promoter (fruP)⁵were found not to enhance cytochromecoxidase expression inR. sphaeroides.An improved cytochromecoxidase purification protocol is reported, which combines histidine elution from a nickel affinity column and anion-exchange chromatography, and results in a higher yield and purity than previously obtained.     

Cloning,Overexpression, Purification and Preliminary Characterization of Human Septin 8

Mammalian septins comprise a family of 14 genes that encode GTP-binding proteins involved in important cellular processes such as cytokinesis and exocytosis. Expression of three different constructs encoding human septin 8 were analyzed and the results show that SEPT8GC, a clone expressing the conserved domain plus C-terminal domain of human septin 8 yields the highest amount of recombinant protein. This protein was purified by affinity chromatography followed by a gel filtration chromatography. CD spectrum of SEPT8GC is characteristic of folded proteins and it presents a transition profile with a T _m of 54 °C. Fluorescence emission spectra, analytic gel filtration and DLS reflect the sample oligomeric heterogeneity with the predominance of dimers in solution. Homology models indicate clearly that the preferred dimer interface is the one comprising the GTP binding site.     

Overexpression,Refolding, Purification of Erbin PDZ Domain from Escherichia coli and Preparation of its Polyclonal Antibody

Abstract

The Erbin was recently identified. The antibody against Erbin has not been commercially available. As a new member of peripheral protein LAP family and novel type of adaptor protein, its functions and binding partners are not completely known. In the present study, cDNA encoding PDZ domain of Erbin was inserted in a prokaryotic expression vector. His-tagged recombinant protein was overproduced in E. coli and purified by Ni-NTA column chromatography. About 14.4 mg of the purified protein was obtained from 500 mL of cell culture. The purity of the recombinant protein was higher than 90%. The polyclonal antibody against this protein was raised. The antibody can recognize both denatured and natural Erbin protein. It will be used to further identify the new binding partners of Erbin and study its unknown functions.     

Cloning, Expression, and Purification of the Uropathogenic Escherichia coli Invasin DraD

In this study we presented a very efficient expression system, based on pET30LIC/Ek vector, for producing DraD invasin of the uropathogenic Escherichia coli and a one-step chromatography purification procedure for obtaining pure recombinant protein (DraD-C-His₆). This protein has a molecular weight of 14,818 and calculated pI of 6.6. It contains a polyhistidine tag at the C-terminus (13 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. Also, we obtained specific antibodies against DraD invasin to develop tools for characterizing the expression and biological function of this protein. The amount and quality of DraD-C-His₆ fusion protein purified from E. coli overexpression system seems to be fully appropriate for crystallographic studies (soluble form), and for establishing role of the protein in bacterium (cultured cell line interaction and in the internalization process) and for obtaining rabbit polyclonal antisera (insoluble form).     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the -factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg^–1 protein which was more than a recombinant P. pastoris GS115 (552 U mg^–1 protein) or KM71H (539 U mg^–1 protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg^–1 protein by P. pastoris GS115, 1176 U mg^–1 protein by P. pastoris KM71H and 1522 U mg^–1 protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 °C) than the wild-type PLC from B. cereus. Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co²⁺ and Mn²⁺ etc., also influenced the activity of the recombinant PLCs.     

ERIC-PCR鉴别苏云金芽胞杆菌与蜡状芽胞杆菌的研究

利用ERIC-PCR技术对苏云金芽孢杆菌(Bt)、蜡状芽孢杆菌(Bc)和对照菌基因组DNA进行扩增,回收、标记BtPCR扩增片段,分别与各菌株的基因组DNA进行斑点杂交和Southern杂交,筛选Bt标识序列。结果显示:Bt各菌株均可扩增得到250bp的特异片段;Bt和Bc均可得到600bp的共有扩增片段;以筛选得到的569bp片段为探针,可特异性地与Bt基因组DNA杂交;ERIC-PCR技术可以在DNA指纹图谱水平区分鉴别Bt与Bc菌,正确反映出两者的亲缘关系。结果表明ERIC-PCR技术在Bt的检测及在Bt与Bc的鉴定中具有较强的实用性。     

棉花磷脂酶C基因的克隆及参与油脂代谢的功能分析

以陆地棉胚珠和纤维为实验材料,利用RT-PCR技术克隆得到磷脂酶C(Phospholipase C,PLC)基因(GhPLC,GenBank登录号:KR154219),将棉花磷脂酶C基因转化拟南芥,分析其在油脂代谢过程中的重要作用。测序鉴定显示,GhPLC基因的全长开放读码框为1 524bp,编码包含508个氨基酸的蛋白质,理论分子量约为55kD。序列比对分析显示,GhPLC属于典型的碱性磷酸酶超家族的磷脂酶。利用pET32a-GhPLC原核表达获得分子量约为55kD左右的重组蛋白GhPLC;酶活力分析显示,重组GhPLC蛋白具有较高的将卵磷脂(PC)催化为二酰甘油(DAG)的酶活力。半定量RT-PCR结果表明,GhPLC基因参与棉花种子和纤维发育过程。构建植物过量表达载体35S∷GhPLC并转化哥伦比亚野生型拟南芥,转基因拟南芥中GhPLC基因的表达和PLC酶活力显著提高,且转基因拟南芥种子的油脂含量提高了5.3%。     

LSECtin-CRD-GST融合蛋白的原核表达、纯化及复性

目的:构建C型凝集素LSECtin主要功能结构域CRD的原核表达载体,在大肠杆菌中表达LSECtin-CRD-GST融合蛋白。方法:根据Gen Bank发布的LSECtin基因序列设计引物,利用基因重组技术将获得的LSECtin-CRDc DNA定向克隆至C端带GST蛋白标签序列的融合表达载体p GEX-6p-1中,转化大肠杆菌Origami(DE3)进行重组蛋白的诱导表达,用GST柱亲和纯化融合蛋白。结果:获得了原核表达载体p GEX-6p-1-LSECtin-CRD,诱导表达出大量相对分子质量约40×103的包涵体融合蛋白,经纯化、复性获得可溶蛋白,经Western印迹鉴定为目的蛋白。结论:获得足量的LSECtin-CRD-GST融合蛋白,为进一步研究CRD蛋白结构域的动态构象变化提供了实验材料。     

Molecular Cloning,Overexpression, and Purification of a Major Xylanase from Aspergillus oryzae

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58°C. It had a Km of 5.1 mg/ml and a Vmax of 123 μmol/min/mg when birch wood xylan was used as a substrate.     

Refolding and Purification of Zymomonas mobilis Levansucrase Produced as Inclusion Bodies in Fed-Batch Culture of Recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE–Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Cloning, Overexpression and Purification of Bacillus subtilis Elongation Factor Tu in Escherichia coli

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His₆ tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

ccpA基因对蜡样芽胞杆菌氨肽酶生产的影响

【目的】构建蜡样芽胞杆菌(Bacillus cereus)ccp A缺失菌株,并初步探索ccp A基因对其碳代谢及氨肽酶生产的影响。【方法】利用温敏型质粒p KSV7构建蜡样芽胞杆菌CZ ccp A基因缺失突变株CZΔccp A,通过回补菌株对敲除株表型进行验证;不同碳源发酵对比菌株碳代谢的变化,进行氨肽酶发酵优化。【结果】成功构建ccp A缺失菌株CZΔccp A与回补菌株CZ1,三株菌在LB培养基中生长无差异;在柠檬酸钠以及甘露低聚糖为碳源时,菌株的代谢产生明显变化;以D-木糖为单一碳源时,氨肽酶的产量提高48.25%。【结论】CZ ccp A基因对柠檬酸钠、甘露低聚糖、D-木糖为单一碳源时的代谢可能具有调控作用,ccp A基因缺失可以提高蜡样芽胞杆菌CZ的氨肽酶产量。     

Construction, Overexpression, and Purification of Arthrobacter globiformis Amine Oxidase–Strep-Tag II Fusion Protein

The copper-containing amine oxidase from Arthrobacter globiformis has been expressed and purified as a fusion protein with a C-terminal Strep-tag II peptide. This tag facilitates the rapid purification of the enzyme on a large scale using the StrepTactin POROS medium. For example, we have demonstrated that 50 mg of protein can be obtained in 2 days from 2 L of Escherichia coli. The purified fusion protein displays turnover and spectroscopic properties that are essentially identical to those of the wild-type enzyme. Given the location of the C-terminus in four amine oxidase crystal structures, this strategy should be quite general for the rapid purification of amine oxidases from multiple sources.