Molecular cloning,expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.     

Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase.

N-Heparan sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the nitrogen of glucosamine in heparan sulfate. The enzyme has been previously purified to apparent homogeneity from rat liver (Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263, 2417-2422). We have now cloned the rat liver enzyme using the following strategy: (a) the amino acid sequence was obtained from tryptic peptides of the purified protein, (b) mixed oligonucleotides were generated based on the sequence of the tryptic peptides, (c) a polymerase chain reaction fragment was obtained using mixed oligonucleotide interprimer amplification of cDNA, and (d) this fragment was used to screen rat liver lambda gt 10 and lambda ZAP libraries. Three clones were obtained, one of which seems to contain the complete coding sequence of the N-heparan sulfate sulfotransferase (N-HSST). Evidence that the cDNA clone corresponds to the previously purified and characterized N-HSST was the following: (a) the predicted sequence of the N-HSST contains all of the 11 tryptic peptides obtained from the purified protein, (b) when a cDNA containing the sequence coding for the N-HSST was introduced in a eukaryotic expression vector and transfected in COS-1 cells, the enzyme activity was expressed 9-fold over controls, and (c) the characteristic of the predicted protein fits with the purified protein in terms of molecular weight, membrane localization, and its being an N-linked glycoprotein. The size of the longest cDNA isolated is 4.1 kilobases, which is in close agreement with the 4.2-kilobase size of one of the mRNA observed in Northern analyses. In addition, messages of 7.0 and 8.5 kilobases were also observed, suggesting that a large portion is untranslated. The latter messages were the major mRNA species detected.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

新生隐球菌MIS1基因的siRNA表达载体的构建及鉴定

目的 构建靶向新生隐球菌MIS1基因的siRNA重组表达载体质粒,并进行鉴定.方法 根据GenBank的MIS1基因序列,按照载体要求设计单链引物,克隆到空载体psilencer4.I-CMV neo中,经过LiAc化学法将重组质粒转染到新生隐球菌细胞中并用G418筛选,利用real time PCR鉴定阳性细胞的MIS1基因水平.结果 重组表达质粒Psilencer4,1-CMV-si-MIS1经PCR、双酶切及测序鉴定,结果证明重组表达载体构建成功,其能在mRNA水平显著抑制MIS1的表达.结论 已成功构建新生隐球菌MIS1基因的siRNA表达载体,为深入研究MIsl在隐球菌相关疾病的发生及发展中的作用提供了技术手段.     

Expression of human thymidylate synthase in Escherichia coli

A cDNA clone encoding thymidylate synthase (TS) has been isolated from a human T-cell library and modified in the 5'-untranslated region to incorporate several unique cloning sites. The gene has been cloned as a cassette into several Escherichia coli expression vectors which did not provide detectable amounts of the enzyme. A successful approach used a constitutive E. coli expression vector developed for the enzyme from Lactobacillus casei. A 115-base pair 5'-untranslated region from the L. casei TS which contains a ribosomal binding site and other regulatory sequences has been fused to the coding region of the human TS gene to provide a construct that is expressed in E. coli. The level of expression was further enhanced by altering the nucleotide sequence of the first 90 base pairs to accommodate common codon use in E. coli. In our best expression system, catalytically active human TS is expressed to a level that represents about 1.6% of the total soluble protein. The recombinant human TS has been purified and characterized; except for the presence of an amino-terminal blocking group, the enzyme has physical and kinetic properties similar to the enzyme isolated from human cells.     

Nucleotide sequence of cDNA clones coding for a brain-type fatty acid binding protein and its tissue-specific expression in adult zebrafish (Danio rerio)

We have determined the nucleotide sequence for two cDNA clones coding for a fatty acid binding protein (FABP) from zebrafish (Danio rerio). Comparison of the sequence with GenBank entries revealed extensive amino acid identity between this zebrafish FABP and brain FABPs (B-FABP) from other species. The zebrafish B-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases BglII or EcoRI suggesting that a single copy of the B-FABP gene is present in the zebrafish genome. Northern blot analysis demonstrated that the zebrafish B-FABP mRNA is approximately 850 nucleotides in length. In situ hybridization revealed that the B-FABP mRNA was expressed in the periventricular gray zone of the optic tectum of the adult zebrafish brain.     

斑马鱼fem-1c cDNA克隆与表达分析

为进一步探究鱼类性别决定的相关机理, 增加对鱼类性控基因表达和功能的认识, 克隆斑马鱼fem-1c 基因并对其进行表达分析。研究采用RACE-PCR方法从斑马鱼卵巢组织cDNA中克隆了fem-1c的cDNA全长序列, 其大小为2701 bp, 编码618个氨基酸。生物信息学分析显示, 斑马鱼FEM-1C蛋白包含9个ANK结构域、2个TPR结构域和2个低复杂性区域, 与其他脊椎动物的FEM-1C蛋白序列保守性较高。脊椎动物的fem-1c与tmed7、trim36等邻近的45个基因具有保守的同线性关系。半定量RT-PCR实验结果显示斑马鱼fem-1c在受精后17d开始表达, 并特异地表达于成体卵巢组织中。RNA原位杂交结果显示, fem-1c基因mRNA定位于卵巢组织的Ⅰ期和Ⅱ期卵母细胞胞质中。fem-1c的时空表达特征暗示其在斑马鱼卵巢分化中具有重要作用。       

Cloning, purification, and properties of Candida albicans thymidylate synthase.

The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIII DNA fragment and was shown to be expressed in a Thy- strain of Escherichia coli. The nucleotide sequence of the TS gene predicted a protein of 315 amino acids with a molecular weight of 36,027. The gene was cloned into a T7 expression vector in E. coli, allowing purification of large amounts of C. albicans TS. It was also purified from a wild-type C. albicans strain. Comparison of several enzyme properties including analysis of amino-terminal amino acid sequences showed the native and cloned C. albicans TS to be the same.     

Efficient synthesis of mouse thymidylate synthase in Escherichia coli

The coding region of the mouse thymidylate synthase (TS)-encoding cDNA (ts) was inserted downstream from the phage T7 promoter and translation initiation signals of the expression vector, pET-3a, and transformed into Escherichia coli BL21(DE3)[pLysS]. When the wild-type (wt) cDNA sequence was used, mouse TS was synthesized in the bacterial cells in response to induction, but the level of expression was low. When the second codon (Leu) was changed from CUG, found in the normal mRNA, to CUU, the level of expression increased 17-fold and TS represented 5-10% of total cell protein. The recombinant enzyme was purified to homogeneity by affinity chromatography. The recombinant TS had the same Mr as the enzyme from cultured mouse fibroblasts. Kinetic studies with the recombinant enzyme showed that the apparent Km values for deoxyuridylate and 5,10-methylenetetrahydrofolate were 10.5 and 22 microM, respectively, which were similar to the values for TS from mouse cell extracts. The mouse ts expression vector will be useful for the large-scale production of the wt enzyme and for the creation and analysis of mutant enzymes by protein engineering techniques.     

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

Nucleotide sequence of a cDNA clone coding for an intestinal-type fatty acid binding protein and its tissue-specific expression in zebrafish (Danio rerio)

We have cloned a cDNA from zebrafish (Danio rerio) that contains an open-reading frame of 132 amino acids coding for a fatty acid binding protein (FABP) of approximately 15 kDa. Multiple sequence alignment revealed extensive amino acid identity between this zebrafish FABP and intestinal-like FABPs (I-FABP) from other species. The zebrafish I-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases PstI Bg/II or EcoRI suggesting that a single copy of the I-FABP gene is present in the zebrafish genome. An oligonucleotide probe complementary to the zebrafish I-FABP mRNA hybridized to an mRNA of approximately 800 bases in Northern blot analysis. In situ hybridization revealed that the I-FABP mRNA was expressed exclusively in the intestine of the adult zebrafish.     

重组斑马鱼CD36蛋白的原核表达及纯化

目的：在大肠杆菌中重组表达斑马鱼CD36蛋白胞外区38～432氨基酸残基段并纯化。方法：PCR扩增斑马鱼CD36蛋白的基因编码区,连接到带有6~His标签的原核表达载体pET-28a中,构建重组表达质粒pET28a-CD36,并转化大肠杆菌BL21（DE3）,用IPTG诱导表达,优化表达条件后用Ni^2＋柱进行纯化。结果：构建了pET28a-CD36重组质粒;目的蛋白在大肠杆菌中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的46．8×10^3。结论：获得了斑马鱼CD36融合蛋白,为其生物学功能研究奠定了基础。     

Cloning and Characterization of Replication Protein A p32 Complementary DNA in Zebrafish (Danio rerio)

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (70, 32, and 14 kDa) that is an essential component of the DNA replication fork. A complementary DNA encoding zebrafish RPA 32-kDa subunit was isolated by screening a zebrafish embryo lambda APII cDNA library with a human RPA p32 cDNA probe. The zebrafish RPA p32 cDNA consisted of 1097 bp encoding 272 amino acid residues. The deduced amino acid sequence shows high similarity to mouse and human RPA p32. In vitro phosphorylation of zebrafish RPA protein by Cdc2 kinase was shown. A recombinant protein of zebrafish RPA p32 containing a short histidine tag at the NH(2)-terminus was overexpressed in Escherichia coli BL21(DE3) pLys using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography. In this article, cloning of the zebrafish RPA p32 cDNA is reported in relation to the study of DNA replication in fish.     

Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli

cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.