产碱性磷酸酶乳杆菌的筛选鉴定、酶的纯化及特性

【背景】碱性磷酸酶(alkaline phosphatase,ALP)是生物体内参与磷酸代谢的调控酶,不同物种的ALP性质与其生理功能有关,提纯后的ALP常用作工具酶,广泛应用于基因工程中,但目前关于乳酸菌中ALP的相关研究甚少。【目的】筛选出一株产ALP且具有潜在益生作用的乳杆菌,对该酶进行分离纯化,并对其性质进行探究,为今后益生菌的开发利用和ALP的工业化生产提供新的微生物资源。【方法】采集蒙古国4个地区的酸马奶样品,通过显色反应初筛和酶活检测复筛对产酶菌株进行筛选,经形态学观察、生理生化鉴定及16S rRNA基因序列同源性比较分析进行菌种鉴定。采用超声破碎法提取ALP,经硫酸铵沉淀、DEAE-52离子交换层析、Sephadex G-200凝胶过滤层析纯化该酶,SDS-PAGE电泳法检测其纯度。【结果】从78株乳酸菌中分离筛选出一株产ALP酶活性最高的乳杆菌(编号为Z23),16S rRNA基因序列长度为1 473 bp,鉴定结果表明为鼠李糖乳杆菌。纯化后的酶比活力为180.27 U/mg,纯化倍数为48.37,酶活回收率为17.05%,该酶亚基相对分子质量为46.7 kD。菌株所产ALP的最适温度为37℃,4℃时酶活最为稳定;最适pH为9.5,在pH 9.0-10.0之间,酶活稳定性可达90%以上;Mg2+和K+对ALP有明显激活作用,Ba2+和Cu2+在低浓度时对ALP有激活作用,高浓度时有抑制作用,Ca~(2+)、Zn~(2+)和EDTA对ALP有强烈的抑制作用。以不同浓度的p-NPP为底物,测得酶的Km值为3.42 mmol/L,Vmax值为1.24 mmol/(L·min)。【结论】本研究对蒙古国地区酸马奶中的益生菌资源有了更为明确的认知,为今后碱性磷酸酶产生菌的筛选和酶的应用开辟了新途径。     

抗伏马菌素单链抗体与碱性磷酸酶的融合表达及活性鉴定

为原核表达抗伏马菌素单链抗体-碱性磷酸酶融合蛋白并分析其活性,本研究根据抗伏马菌素单链抗体H2的基因序列设计引物,PCR扩增获得目的基因,经限制性核酸内切酶SfiⅠ和Not Ⅰ的酶切位点克隆到pDAP2/S载体中,转化大肠杆菌(Eschrichia coli)菌株XL1-Blue并鉴定阳性转化子.IPTG诱导H2-AP...     

家蚕蛋白酪氨酸磷酸酶基因克隆、表达纯化与结构分析

蛋白酪氨酸磷酸酶(Protein tyrosine phosphatase,PTP,EC 3.1.3.48)特异性地催化去除磷酸化修饰的酪氨酸残基上的磷酸基团,导致蛋白去磷酸化,从而调控了细胞生长、增殖、分化和免疫等生命活动。家蚕Bombyx mori蛋白酪氨酸磷酸酶h(BmPTP-h)参与了核型多角体病毒(Nucleopolyhedrovirus,NPV)在家蚕体内的复制过程,但目前对于BmPTP-h结构和性质的了解并不多。本文从家蚕中肠克隆了BmPTP-h基因编码序列,分析了BmPTP-h的基因组结构、mRNA结构、序列特征、二级结构和溶液中的状态。同源氨基酸序列比对分析表明BmPTP-h与多种昆虫NPV的PTP序列具有高相似度,暗示了它们可能具有共同的起源和相似的功能。文中构建了原核表达载体,通过大肠杆菌在25℃下表达获得了可溶性的重组BmPTP-h,利用Ni-NTA亲和层析纯化了BmPTP-h。凝胶过滤分析显示BmPTP-h在溶液中可以形成聚集体和单体。圆二色光谱分析显示重组的BmPTP-h包含α螺旋结构,升高温度导致BmPTP-h的α螺旋结构去折叠,α螺旋结构含量下降。这些研究为深入研究BmPTP-h的结构和调控机理提供了基础。     

内毒素结合肽的原核表达、纯化及生物学活性鉴定

重组人内毒素结合肽 (endotoxinbindingpeptide ,EBP)融合蛋白在大肠杆菌中表达 ,分离和纯化后对其进行生物学活性观察 .将构建好的PinpointⅩa3 EBP生物素融合表达载体转化大肠杆菌DH5α ,IPTG诱导表达菌株 ,亲和层析法纯化表达产物 ,因子Ⅹa(factorⅩa)切割分离内毒素结合肽 ,采用凝胶过滤和反相液相高效色谱法两步纯化 ,从相对分子质量、N端 1 0个氨基酸的序列分析等方面进行鉴定 ;利用人单核细胞U937对重组内毒素结合肽进行了生物学活性的检测 .结果发现 ,内毒素结合肽以包涵体形式存在 ,因子Ⅹa酶切融合蛋白后得到 3 5kD的内毒素结合肽 ,纯化后内毒素结合肽纯度达 99%以上 ,N端 1 0个氨基酸的分析结果与预期相符 ;初步证实内毒素结合肽具有较好的LPS结合活性 ,能够抑制LPS的作用 .经原核表达及纯化复性 ,获得了具有较好生物学活性的内毒素结合肽 ,为进一步研究其功能奠定了良好的基础     

尿酸氧化酶在大肠杆菌中的表达、纯化及活性鉴定

尿酸氧化酶(urate oxidase,Uricase,EC.1.7.3.3)是一种能将尿酸氧化为尿囊素的蛋白酶。合成黄曲霉(Aspergillus flavus)尿酸氧化酶基因,构建表达载体pET43.1a/uox,重组质粒经双酶切鉴定和序列分析,证明插入序列正确,转化到大肠杆菌(Escherichia coli)JM109,菌株经诱导表达尿酸氧化酶蛋白,目的蛋白经过超声破碎,经检测以可溶性蛋白为主;菌体经超声破碎后,上清经过阴离子柱和阳离子柱两步纯化,得到尿酸氧化酶纯品,纯品以分光光度法进行体外酶活性测定。结果显示:尿酸氧化酶在大肠杆菌中获得高效表达,目的蛋白占菌体总蛋白的50%;表达产物经过两步层析柱纯化,获得电泳扫描纯度为95%的纯品;在体外活性测定中具有分解尿酸的能力,在临床检测和治疗中有重要意义。     

SUMO蛋白酶活性片段的表达、纯化及活性测定

利用PCR技术人工合成编码酿酒酵母泛素样特异性蛋白酶1 (Ubiquitin-like specific protease 1,Ulp1)第403到621个氨基酸残基之间的DNA片段Ulp1p,并连接到大肠杆菌表达载体pET-3c中,构建出重组表达质粒pET-Ulp1p。将重组质粒转化至大肠杆菌BL21(DE3)中,氨苄青霉素抗性筛选转化子。经IPTG诱导4h后, SDS-PAGE结果显示,Ulp1p为可溶性表达,表达量占菌体总蛋白的50.8%。通过Ni-NTA凝胶亲和层析和G-25凝胶层析联用可以获得纯度大于95%的Ulp1p。Western-blotting分析表明,Ulp1p能与6xHis抗体产生免疫反应。以重组蛋白SUMO-hEGF(人表皮生长因子)和GST-SUMO-MT(金属硫蛋白)为底物进行酶切分析,结果显示,Ulp1p能特异性水解这两种SUMO融合蛋白,其比活为1.386 x104U/mg。     

重组内肽酶AspN的原核表达纯化和活性鉴定

蛋白内肽酶AspN是一种锌金属内切肽酶,能选择性切割天冬氨酸的N端肽键,广泛应用于蛋白的多肽制备及质量肽图谱鉴定。目前内肽酶AspN来源于细菌分泌,产量低,制备困难,成本高,极大地限制了该酶的应用。将脑膜脓毒性黄杆菌分泌的蛋白内肽酶Asp N所对应的基因克隆入表达载体p ET32a,导入E.coli BL21(DE3),首次运用原核表达系统进行可溶性融合表达,亲和层析对重组蛋白进行纯化。用HPLC、SDS-PAGE和荧光底物Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe(NO2)-Tyr-Asp对重组酶进行酶活鉴定。结果表明重组的内肽酶Asp N具有与标准品基本一致的酶切活性,能够较好地应用于生物和制药领域。     

重组人RGD-sTRAIL的表达、纯化及抗肿瘤活性研究

利用PCR技术构建RGD短肽与人肿瘤坏死因子凋亡配体(胞外区114-281)的融合基因,将该DNA片段克隆到原核表达载体pET-11a中。重组质粒转化大肠杆菌BL21(DE3),IPTG诱导后可表达相对分子质量约为20000的目的蛋白,占菌体蛋白的20％左右,且大多数重组蛋白以不溶的包涵体形式存在。Western印迹表明目的蛋白具有人sTRAIL的抗原性。表达产物经变性、复性、离子交换层析和分子筛等步骤,可以得到纯度大干95％的RGD-sTRAIL重组蛋白。肿瘤细胞体外实验发现,纯化后的RGD-sTRAIL重组蛋白能明显抑制人肺癌细胞A549生长,并呈剂量依赖性。研究结果表明,通过复性,包涵体中的RGD-sTRAIL蛋白得到正确的折叠,并在体外具有杀伤肿瘤细胞的活性,从而为进一步研究体内靶向性杀伤肿瘤细胞奠定了基础。     

假单胞菌株M18 gacA基因的克隆、表达及蛋白纯化

GacA是GacS/A双元调控系统的一个组分, 克隆假单胞菌株M18中的gacA基因。测序结果同Pseudomonas sp. PAO1比较, 该基因2个碱基的改变未引起氨基酸的改变。将该基因克隆到表达质粒pET28b (+)中, 将重组质粒经热激转化至大肠杆菌BL21 (DE3)中, IPTG诱导表达。表达产物经金属螯合层析柱纯化, 蛋白纯度约为98%, 质谱鉴定证明纯化的蛋白为GacA蛋白。CD谱分析GacA包含了4% α-helix, 48% β-sheet 和48%无规则卷曲。GacA蛋白的获得为进一步研究其晶体结构、生物学性能以及GacS/A双元调控系统奠定了基础。     

Purification and Characterization of Glutamate 1-Semialdehyde Aminotransferase from Barley Expressed in Escherichia coli

The immediate precursor in the synthesis of tetrapyrroles is Δ-aminolevulinate (ALA). ALA is synthesized from glutamate in higher plants, algae, and certain bacteria. Glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8) (GSA-AT), the third enzyme involved in this metabolic pathway, catalyzes the transamination of GSA to form ALA. The gene encoding this aminotransferase has previously been isolated from barley (Hordeum vulgare) and inserted into an Escherichia coli expression vector. We describe herein the purification of this recombinant barley GSA-AT expressed in Escherichia coli. Coexpression of GroEL and GroES is required for isolation of active aminotransferase from the soluble protein fraction of Escherichia coli. Purified GSA-AT exhibits absorption maxima characteristic of vitamin B₆-containing enzymes. GSA-AT is primarily in the pyridoxamine form when isolated and can be interconverted between this and the pyridoxal form by addition of 4,5-dioxovalerate and 4,5-diaminovalerate. The conversion of GSA to ALA under steady-state conditions exhibited typical Michaelis-Menten kinetics. Values for K_m (d,l-GSA) and k_cat were determined to be 25 micromolar and 0.11 per second, respectively, by nonlinear regression analysis. Stimulation of ALA synthesis by increasing concentrations of d,l-GSA at various fixed concentrations of 4,5-diaminovalerate supports the hypothesis that 4,5-diaminovalerate is the intermediate in the synthesis of ALA.     

人Leptin基因在大肠杆菌中的高效表达、纯化及其生物学活性研究

将人Leptin表达质粒pBV220-OB转化E.coliJM109,经热诱导获得了目的蛋白的表达。经SDS-PAGE鉴定分析,表达产物以包涵体形式存在,目的蛋白表达量占菌体总蛋白的40%以上。通过包涵体分离,Sephacryl S200HR凝胶和DEAE52离子交换层析及Hypersil C18柱反相色谱纯化,获得纯度在95%以上,内毒素含量小于10EU/mg的高纯度的重组人Leptin。Western-blot鉴定表明,纯化表达产物能和抗Leptin抗体特异性结合;蛋白质N端15个氨基酸序列分析结果和预期的序列一致。纯化产物经复性处理,其分子中Cys96和Cys146形成二硫键。体内活性检测显示,纯化和复性的rh-Leptin明显抑制BALB/c小鼠的进食和体重增长,提示其具有明显的生物学活性。     

Purification and Characterization of L-2,3-Butanediol Dehydrogenase of Brevibacterium saccharolyticum C-1012 Expressed in Escherichia coli

The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was mesured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.     

大肠杆菌梭曼水解酶的纯化和性质

大肠杆菌梭曼水解酶的纯化和性质邵煌,刘昌玲,肖美珍,孙曼霁（北京军事医学科学院毒物药物研究所,北京１００８５０）梭曼属Ｇ类神经性有机磷毒剂．自然界发现多种细菌中均存在梭曼水解酶（Ｓｏｍａｎａｓｅ）活性［１－３］．研究细菌梭曼水解酶,寻求生物解毒的方法...     

Purification and Characterization of Escherichia coli MreB Protein

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm.     

Purification and Characterization of Glucokinase in Escherichia coli B

Glucokinase was purified from Escherichia coli B cells dosed with a hybrid plasmid carrying the gene for glucokinase. The enzyme was purified about 170-fold and was homogeneous on polyacrylamide gel electrophoresis. The enzyme was 49,000 in molecular weight and consisted of two subunits having a molecular weight of 24,500. The glucokinase catalyzed phosphorylation of D-glucose, D-mannose, D-glucosamine, and 2-deoxy-D-glucose, consuming ATP as a phosphoryl donor. Besides ATP, other nucleoside triphosphates such as ITP, GTP and UTP were also utilized as phosphoryl donors. The enzyme required free sulfhydryl groups and Mg²⁺ for activity. Other properties of the glucokinase were characterized and compared with those of glucokinases from various sources.     

Expression,Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase.     

大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子的纯化

对重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）高效表达克隆ｐＺＷ．ＧＭ的表达产物进行了纯化,并对纯化的ＧＭ－ＣＳＦ进行了Ｎ端氨基酸序列分析。人ＧＭ－ＣＳＦ基因表达产物在大肠杆菌中以不溶性包涵体形式存在,经过超声破菌、包涵体抽提、凝胶过滤层析、复性、离子交换一系列化步骤,终产物纯度达９９％,按蛋白总量计算回收率达１０％,比活性达１×１０＾７ｕ／ｍｇ蛋白质。通过测定纯化人ＧＭ－ＣＳＦ的Ｎ端１     

大鼠α-酰胺化酶(α-AE)在大肠杆菌中的表达、纯化及活性研究

α-酰胺化是神经和内分泌系统中许多生物活性肽重要的翻译后加工过程,C末端α酰胺基团的存在对于许多生物活性肽的生物活性极为重要。本研究通过PCR扩增获得了编码大鼠α-酰胺化酶的基因,以pET-30a为载体,重组质粒pET-A转化至 E.coli BL21成功表达了α-酰胺化酶。采用Ni2+-NTA亲和层析纯化重组蛋白。该蛋白具有催化三肽底物((Dns-Tyr-Val-Gly)成为酰胺化二肽 (Dns-Tyr-Val-NH2)的酶活性,这表明重组蛋白是α-酰胺化酶,有可能用于生物活性肽的酰胺化研究。