Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578

Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity.     

Klebsiella pneumoniae yggG Gene Product: A Zinc-Dependent Metalloprotease

Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.     

Antimicrobial Activities of Hexane Extract and Decussatin from Stembark Extract of Ficus congensis

Ficus congensis (Moraceae) is used traditionally in the treatment of various diseases including infectious diseases, infertility, and gastrointestinal disorders. Investigation of hexane extract of the stem bark using chromatographic techniques led to isolation of a xanthone, 1-hydroxy-3,7,8-trimethoxyxanthone (Decussatin). The compound was elucidated based on spectroscopic methods such as nuclear magnetic resonance (NMR), UV, IR, and mass spectrometry (MS). Decussatin and the hexane extract were screened in vitro for antibacterial and antifungal activities using broth microdilution (MHB) and disc Agar diffusion (DAD) techniques against Escheichia coli, Bacilus substilis, Klebsiela pneumonia, Staphylococcus aureus, Aspergillus fumigatus, Trichophyton mentagrophytes, Trichophyton rubrum and Candida albicans. Hexane extracts showed potent antibacterial activity against E. coli and B. subtilis with minimum inhibitory concentrations (MIC) of 8 mg/mL and 5 mg/mL, respectively, while Decussatin of the highest concentration (8 mg/mL) used in this study showed no appreciable antimicrobial activity. Only hexane extract was active against C. albicans with a MIC of 1 mg/mL.     

Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.     

体外拼装苦瓜降糖多肽P基因

目的 以苦瓜降糖多肽P基因为例,探讨用寡核苷酸片段体外拼装基因的方法。方法根据已知苦瓜降糖多肽P的蛋白序列和大肠杆菌的密码子偏爱性,反向翻译出可在大肠杆菌内表达的苦瓜降糖多肽P基因,设计24条40 bp的降糖多肽P基因寡核苷酸片段引物,通过PCR进行寡核苷酸片段的扩增和拼装。将拼装好的多肽P基因的cDNA与pET-32a载体连接后,转化E.coli BL21(DE3),IPTG诱导表达,表达产物经Ni2+-NTA纯化后检测其对四氧嘧啶引起的糖尿病小鼠的降糖活性。结果苦瓜降糖多肽P基因经2轮PCR扩增后,即可见目的 条带;经第3轮PCR扩增,即可见清晰的目的 条带。经测序鉴定,与设计的降糖多肽P基因序列完全一致。重组蛋白在E.coli BL21(DE3)中获得可溶性表达,且可与鼠抗His单抗发生特异性反应。纯化的融合蛋白纯度达90%以上,产量为10 mg/L,其对四氧嘧啶引起的糖尿病小鼠具有降糖活性。结论体外基因拼装法是一种有效获取目的 基因的方法,可进行密码子偏爱性设计,在宿主系统中有效表达目的 基因。     

肠道病毒71型3C基因原核表达质粒的构建表达及纯化

目的构建肠道病毒71型(Entervirus71,EV71)3C基因的原核表达质粒,在大肠杆菌中进行表达并纯化。方法以病毒的反转录产物为模板,PCR扩增EV71 3C基因,克隆至pGEx-4T-1载体中,构建重组表达质粒pGEx-3C,转化大肠杆菌BL21(DE3)中,IPTG诱导表达。表达的重组融合蛋白GSR-3C经GST-Agarose亲和层析和分子筛层析纯化后,Western blot检测其反应原性。结果重组表达质粒pGEx-3C经双酶切及测序证实构建正确;表达的重组融合蛋白相对分子质量约46 000,表达量约占菌体总蛋白的50%,主要以可溶性形式表达;纯化后的重组融合蛋白纯度大于90%,可与小鼠抗EV71单克隆抗体特异性结合。结论成功构建了重组表达质粒pGEx-3C,在大肠杆菌中高效分泌表达并纯化了重组GST-3C融合蛋白,为EV713C蛋白酶结构与功能的研究及3C靶向药物和疫苗的研究奠定了基础。     

无乳链球菌GapC蛋白和鼠伤寒沙门菌鞭毛蛋白的融合表达及纯化

目的构建无乳链球菌(Streptococcus agalactiae,S.agalactiae)gapC基因和鼠伤寒沙门菌(Salmonella typhimu-rium,S.typhimurium)fliC基因的融合基因gapC-fliC,并在大肠杆菌中表达融合蛋白。方法应用PCR技术分别扩增鼠伤寒沙门菌鞭毛蛋白fliC基因和无乳链球菌gapC基因片段,通过柔性肽(Gly4Ser)2编码序列将二者串联并克隆至质粒pQE-30上,构建重组原核表达质粒fliC-gapC-pQE30,转化E.coli XL1-Blue,IPTG诱导表达,并进行SDS-PAGE分析。表达产物经镍离子亲和层析柱纯化后,进行Western blot分析及融合蛋白活性检测。结果重组表达质粒fliC-gapC-pQE-30经双酶切和测序证明构建正确;表达的融合蛋白FliC-GapC相对分子质量约95 000,表达量占菌体总蛋白的58%,主要以包涵体形式存在,且可与小鼠抗鼠伤寒沙门菌和抗无乳链球菌多抗血清发生特异性反应,具有较好的甘油醛-3-磷酸脱氢酶(GAPDH)活性。结论成功构建了重组表达质粒fliC-gapC-pQE-30,并在E.coli XL1-Blue中表达了融合蛋白,为下一步动物免疫保护性试验的研究奠定了基础。     

cTnI特殊位点抗原的重复表达、免疫及其抗体效价检测

构建了含人cTnI蛋白稳定区段2个特殊抗原决定簇位点的pMAL-Antigen质粒. 利用BamHI/BgLII两限制性内切酶的同尾酶作用进行连接,在pMAL-p2x质粒的MBP(麦芽糖结合蛋白)之后接入了上述5个相同的片段. 此质粒在大肠杆菌E. coli BL21 (DE3)中经1.0 mmol/L IPTG诱导表达出的融合蛋白MBP-Antigen经Amlyose-resin亲和层析柱纯化后免疫BABL/C小鼠,成功获得了能抗人cTnI和抗牛cTnI的抗血清.     

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C(10)), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the K(m) values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.     

嗜人类T淋巴细胞病毒Ⅰ型核心蛋白P24基因的克隆与表达

目的 从感染HTLV-1的中国患者外周血单核细胞（PBMCs）中扩增编码HTLV-1核心蛋白P24的cDNA,并在大肠杆菌中进行表达。方法 提取感染者PBMCs基因组DNA,应用巢式PCR方法扩增出HTLV-1核心蛋白P24的cDNA并测序,与pGEX-20T载体构建重组质粒,在大肠杆菌BL21中表达,采用ELISA和Westernblot分析重组蛋白活性。结果HTLV-1核心蛋白P24基因序列高度保守,构建重组载体后,在大肠杆菌中表达的重组蛋白,经检测具有较强的抗原活性。结论 成功地表达了HTLV-1型病毒的核心蛋白P24基因,为国产诊断试剂和疫苗的研发打下了基础。     

Development of silk-like materials based on Bombyx mori and Nephila clavipes dragline silk fibroins

In order to develop new silk-like materials in the form of fiber and non-woven nano-fiber, this study synthesized a new silk-like protein by selecting the sequence from the crystalline region of Bombyx mori silk fibroin, (GAGSGA)₆, to imitate the processing condition of the silk fibroin with the combination of the sequence YGGLGSQGAGRG, the hydrophilic motif of spider dragline silk which was considered as the origin of supercontraction of spider dragline silk (Yang Z, et al. J Am Chem Soc 2000;122: 9019-25). The CD pattern of the silk-like protein in hexafluoroacetone (HFA) indicates that it takes helical structure. The solid-state structures of the silk-like protein before and after methanol treatment were determined to be random coil and Silk II structure (mainly β-sheet structure), respectively, using ¹³C CP/MAS NMR. The ¹³C CP/MAS NMR spectra of the protein after methanol treatment in hydrated state showed that the random coil peaks from Ala Cβ and Ser Cβ carbons become sharper compared with the corresponding peaks in the dry state. The 2D-WISE spectra in the hydrated state also showed increase of the narrow components for these carbons. Thus, water molecules are relatively easy to access at the random coil regions of the protein. The fiber formation of the silk-like protein was possible with wet-spinning or electrospinning methods using HFA as dope solvent and methanol as coagulation solvent.     

Study of Linkage between Glutathione Pathway and the Antibiotic Resistance of Escherichia coli from Patients’ Swabs

In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase—GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (K_m 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of “more resistant” E. coli exhibited differences in K_m between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group.     

HIV-1C亚型调控蛋白Nef在大肠杆菌中的表达

目的表达人免疫缺陷病毒1型C亚型调控蛋白Nef,为研制HIV疫苗寻找新的途径。方法通过PCR扩增,获得HIV-1C亚型Nef基因片段,并将其克隆到原核表达载体pGEX-5X-1中,转化大肠杆菌BL21(DE3),IPTG诱导表达后,以SDS-PAGE和Westernblot分析表达产物。结果表达产物是相对分子质量为50000的GST融合蛋白,且能与p27单克隆抗体发生特异性反应。结论重组Nef蛋白在大肠杆菌中得到了有效表达。     

人内毒素结合肽基因的突变及突变体蛋白表达

目的分析亲代人内毒素结合肽一级结构,选择可能影响其生物学活性的氨基酸对应碱基位点进行突变,克隆基因突变体mEBP基因并研究其蛋白表达。方法以亲代EBP基因为基础,结合DNASIS软件理化性质分析确定突变碱基,应用PCR定点诱变技术进行第5、18位谷氨酰胺→赖氨酸的定点突变。并将其克隆至原核融合表达载体pinpointXa-3,转化大肠杆菌BL21(DE3)pLysS进行表达,经Western blot鉴定。结果突变EBP基因13及52位核苷酸由C突变为A,构建的阳性重组子经酶切及测序鉴定证实与设计序列完全一致,经IPTG诱导在大肠杆菌BL21(DE3)pLysS表达生物素化的融合蛋白,Western blot鉴定显示能与抗生物素单克隆抗体结合。结论已成功获得EBP基因突变体,并在大肠杆菌以融合蛋白的形式进行表达。     

Partial (13)C and (15)N chemical-shift assignments of the disulfide-bond-forming enzyme DsbB by 3D magic-angle spinning NMR spectroscopy

DsbB is a 20 kDa Escherichia coli inner-membrane protein that catalyzes disulfide-bond formation in periplasmic proteins. We report highly resolved, multidimensional magic-angle spinning NMR spectra at 750 MHz (1)H frequency, which enable partial (13)C and (15)N chemical-shift assignments of the signals. The narrow line widths observed indicate excellent microscopic order of the protein sample, suitable for full structure determination by solid-state NMR. Experiments were performed exclusively on uniformly (13)C,(15)N-labeled DsbB. Chemical-shift-correlation experiments based on dipolar transfer yielded strong signals in the 3D spectra, many of which have been site-specifically assigned to the four transmembrane helices of DsbB. Significant numbers of additional residues have been assigned to stretches of amino acids, although not yet placed in the amino acid sequence. We also report the temperature dependence of signal intensities from -50 degrees C to 0 degrees C, a range over which samples of DsbB are highly stable. Structural and dynamic information derived from SSNMR studies can give insight into DsbB in a state that so far has not been successfully crystallized.     

鸡IL-18成熟蛋白基因的克隆及在大肠杆菌中的表达

目的　以鸡脾细胞mRNA为模板扩增编码鸡IL 18成熟蛋白的cDNA ,并在大肠杆菌中进行表达。方法　用PHA和LPS激活的AA肉鸡脾细胞 ,提取mRNA ,以RT PCR法扩增出编码鸡IL 18成熟蛋白的cDNA并测序 ,与pET 2 8b载体构建重组质粒 ,并在大肠杆菌BL2 1(DE3)plysS中表达 ,用SDS PAGE检测表达产物。结果　所克隆的核苷酸片段包含了鸡IL 18全部成熟蛋白编码基因 ,其大小为 5 0 7个核苷酸 ,编码 16 9个氨基酸 ;构建的重组表达载体在大肠杆菌BL2 1(DE3)plysS中表达出相对分子质量为 190 0 0的重组蛋白。结论　已成功克隆和表达了鸡IL 18成熟蛋白基因。     

颗粒裂解肽G13结构域的表达及其对大肠杆菌活力的影响

目的构建携带颗粒裂解肽G13结构域的原核表达质粒,在大肠杆菌BL21(DE3)中表达目的蛋白,并检测其对大肠杆菌活力的影响。方法人工合成G13结构域编码DNA序列,PCR扩增后,用T-A克隆法与pBAD/TOPOThioFusion表达载体连接。通过PCR鉴定及测序筛选阳性重组质粒,转化大肠杆菌BL21(DE3),经阿拉伯糖诱导表达,SDS-PAGE检测表达情况,同时监测诱导表达前后工程菌A600值的变化及菌液中的突变体。结果工程菌经诱导,表达出相对分子质量约15000的特异蛋白,表达量占菌体总蛋白的3%左右。随着外源蛋白的表达,菌液A600值下降,随后又缓慢上升。提取的质粒测序结果表明,其启动子的-35区和-10区均发生了突变,随着诱导时间的增加,突变细胞的比例不断增加。结论已成功构建了G13结构域原核表达质粒,并在大肠杆菌中表达出G13融合蛋白。该异源蛋白的表达导致工程菌的活力降低。     

A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.     

小鼠白细胞介素-18的克隆及在原核细胞中的表达

目的克隆小鼠白细胞介素-18(mIL-18)全长cDNA,并使其在大肠杆菌中表达。方法利用RT-PCR和巢式-PCR,从活化小鼠腹腔巨噬细胞中扩增成熟IL-18的全长cDNA。经双酶切,将该cDNA片断插入表达载体pRSET-C,构建含T7启动子的原核表达载体pRSET-mIL-18。测序鉴定后,将重组体转化大肠杆菌BL21(DE3),在IPTG和/或乳糖的诱导下,使重组质粒获得表达。结果重组质粒读码框及mIL-18的序列与预期一致,表达产物经SDS-PAGE和Western blot分析获得证实。结论已成功构建了原核表达载体pRSET-mIL-18,并使其在大肠杆菌中获得了融合表达。     

Protein interactions in the Escherichia coli cytosol: an impediment to in-cell NMR spectroscopy

Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15)N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein "stickiness" is a limiting factor in the application of in-cell NMR spectroscopy.