酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

家蚕浓核病毒(镇江)株主要结构蛋白基因的克隆及表达

家蚕浓核病毒(Bombyx mori densovirus,BmDNV)是一种昆虫细小病毒.与其它昆虫细小病毒感染昆虫体内多种组织不同,家蚕浓核病毒只感染家蚕中肠上皮组织的圆筒型细胞,感染该病毒细胞的细胞核可以被孚尔根和甲基绿浓染,在病毒感染的早期中肠上皮组织细胞数量增加,形成褶皱,最后感染细胞脱落到肠腔中[1-3].自从20世纪70年代末日本学者证实家蚕浓核病是由于家蚕浓核病毒感染引起的以来[4],已经分离得到了多个病毒株系[5-8].根据它们在血清学、理化特性、品种感受性和病理特征等方面的差异,分为BmDNV-1(伊那株)和BmDNV-2(以山梨株为代表)[8-11].     

Biochemical and genetic analyses of a novel gamma-cyclodextrin glucanotransferase from an alkalophilic Bacillus clarkii 7364

On screening for microorganisms in soil obtained in Japan that produce large amounts of gamma-cyclodextrin (gamma-CD), we identified a novel alkalophilic bacterium, Bacillus clarkii 7364. The cyclodextrin glucanotransferase (CGTase) secreted into the culture medium by this bacterium was purified by affinity chromatography on a gamma-CD-immobilized column, followed by chromatography on a gel filtration column. The enzyme converted 13.7% of pre-gelatinized potato starch (10% w/w per reaction mixture) into CDs, and the majority (79%) of the product CDs was of the gamma form. This property is quite unique among known CGTases and thus we named this enzyme gamma-CGTase. The N-terminal and internal amino acid sequences of gamma-CGTase were determined and used to design PCR primers for amplification of the nucleotide sequence that encodes the gamma-CGTase gene. The entire gene sequence amplified by PCR was determined and then cloned into E. coli. The recombinant enzyme synthesized by E. coli retained biochemical properties quite similar to those of the original one. Comparison of the deduced amino acid sequence of gamma-CGTase with those of other known CGTases that have different product specificities revealed the importance of subsites -3 and -7 for the preferential gamma-cyclization activity.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.     

Controlled specific expression and purification of 6×His-tagged proteins in Pseudomonas

The Coxiella burnetii icd gene encoding an immunogenic dimeric NADP(+)-dependent isocitrate dehydrogenase (IDH) was cloned by screening a C. burnetii genomic library with a human positive serum and sequenced. The predicted gene product consists of 427 amino acids (M(r) = 46,600) and showed high identity to the IDHs of Escherichia coli (74%), Salmonella enterica (73%) and IDH-I of Vibrio sp. (71%). The cloned gene complemented an icd-defective E. coli mutant producing a recombinant IDH that had the same biochemical properties as the enzyme from purified C. burnetii. Unlike the homologs from other bacteria, the cloned enzyme was expressed to the highest level in low pH conditions. This distinct property of the cloned IDH suggests that C. burnetii icd gene may have a role in the adaptation of the organism to the harsh acidic environment of the eucaryotic phagolysosomes.     

Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product

The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.     

Chemical synthesis and expression of a synthetic gene for the flavodoxin from Clostridium MP

A gene coding for the flavodoxin from Clostridium MP was designed, synthesized, and expressed in Escherichia coli. The sequence of the coding region was derived from the published amino acid sequence of the protein (Tanaka, M., Haniu, M., Yasunobu, K.T., and Mayhew, S. G. (1974) J. Biol. Chem. 249, 4393-4397) and was designed for optimal expression and for use of the cassette mutagenesis approach. The structural gene was subassembled in three sections, each of which was constructed by the enzymatic ligation of three complementary pairs of chemically synthesized oligodeoxyribonucleotides having short single-stranded ends complementary to that of the adjacent pair. Coligation of the three sections produced the final structural gene which consists of 420 nucleotides. The synthetic gene was cloned behind the hybrid tac promoter (Amman, E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) in the pKK223-3 vector or adjacent to the strong T7 RNA polymerase promoter in the pET-3a expression vector (Rosenberg, A.H., Lade, B. N., Chui, D-S., Lin, S-W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56, 125-135) for expression in E. coli. Upon induction with isopropyl-beta-D-thiogalactoside, the flavodoxin polypeptide was expressed from the artificial gene to levels approaching 20% of total extractable proteins using either expression system. The flavodoxin was purified from cellular extracts as the holoprotein containing bound flavin mononucleotide. The recombinant flavodoxin protein was found to have an ultraviolet/visible spectrum, amino-terminal sequence, and amino acid composition identical to the wild-type flavodoxin protein purified from Clostridium MP. This work represents the first chemical synthesis and expression in E. coli of an artificial gene coding for a bacterial flavodoxin.     

Sequence analysis of nutA gene encoding membrane-bound Cl(-)-dependent 5'-nucleotidase of Vibrio parahaemolyticus

The membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus is unique in requiring Cl- for activity. We cloned the nutA gene encoding the 5'-nucleotidase and sequenced it. It contained an open reading frame consisting of 1,680 nucleotides capable of encoding a protein of 560 amino acid residues. The first 21 amino acid residues of the N-terminal portion of this protein seem to be a signal peptide. The rest of the polypeptide (539 residues) is hydrophilic, and its molecular weight was calculated to be 60,008, which is in good agreement with the value of 63 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the 5'-nucleotidase derived from the cloned nutA gene. We tried to determine the amino acid sequence of the N-terminal portion of the purified enzyme. However, the N-terminal residue seemed to be blocked. As this 5'-nucleotidase can be solubilized from membrane vesicles with detergent, it may be a lipoprotein. The amino acid sequence around the possible cleavage site of the 5'-nucleotidase had homology with the sequences of the cleavage sites of the lipoproteins of Escherichia coli and other bacteria. The amino acid sequence had high (about 60%) homology with the sequence of periplasmic 5'-nucleotidase (uridine diphosphate sugar hydrolase, the product of the ushA gene) of E. coli. It also contained regions that showed some homology with the nucleotide binding sites of many nucleotide binding proteins.     

Cloning and sequencing of the beta-fructofuranosidase gene from Bacillus sp. V230.

The beta-fructofuranosidase gene (bff) from Bacillus sp. V230 has been cloned in Escherichia coli and its nucleotide sequence has been analyzed. The product of bff consists of a signal sequence of 32 amino acid (a.a.) residues for secretion and 455 a.a. residues of the extracellular beta-fructofuranosidase. The a.a. sequence of the bff product has similarities with those of the Bacillus subtilis levanscrase (63.7% identity), the Streptococcus mutans fructosyltransferase (33.7%), and the Zymomonas mobilis levanscrase and beta-fructofuranosidase (15%).     

Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene

Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein. The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues. The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein. When the cloned gene was expressed in E. coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800). The product was labeled with [3H]glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase. We thus designed the gene product as lipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane. A possible orientation of lipoprotein-28 in the E. coli envelope is discussed.     

Cloning,Sequencing, and Expression of the Gene Encoding an Intracellular β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular β-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50°C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with β-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with β-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50°C.     

Micrococcus luteus homolog of theEscherichia coli uvrA gene: Identification of a mutation in the UV-sensitive mutant DB7

Summary Restriction fragments ofMicrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in anEscherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation. Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination. The putative product of the open reading frame shares an extensive amino acid sequence homology with theE. coli UvrA protein comprising 940 residues. The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of theM. luteus protein. In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. These results indicate that the gene defined by the mutation in DB7 represents a homolog of theE. coli uvrA gene. Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to theE. coli UvrABC excinuclease. Dedicated to the memory of Shunzo Okubo (1930–1978), who considered the possibility of a double-mutant status for the mutant DB7 most seriously     

Sequence of the phosphomannose isomerase-encoding gene of Salmonella typhimurium

The pmi gene, encoding phosphomannose isomerase, of Salmonella typhimurium, was cloned in Escherichia coli K-12, and the protein product visualised in minicells. The cloned gene was sequenced; there was 77.4% nucleotide homology between the cloned pmi gene and the analogous manA gene of E. coli K-12, and 86.2% amino acid sequence homology between their presumptive gene products.     

Nucleotide sequence and expression of the cloned gene of bacteriophage SP6 RNA polymerase.

The coding region of the gene for bacteriophage SP6 RNA polymerase was cloned into pBR322, and its entire nucleotide sequence was deduced. The predicted amino acid sequence for the polymerase consists of 874 amino acid residues with a total molecular weight of 98,561 daltons. Comparison of the amino acid sequence with that of T7 RNA polymerase reveals that regions with partial homology are present along the sequence. The coding region of SP6 RNA polymerase was inserted into an E. coli expression vector. The polymerase gene was efficiently expressed in E. coli cells, and the enzymatic properties of the expressed polymerase were very similar to those of the enzyme synthesized in SP6 phage-infected Salmonella typhimurium cells.     

Cloning and analysis of the gene (rpoDA) for the principal sigma factor of Pseudomonas aeruginosa

A gene (rpoDA) of Pseudomonas aeruginosa whose gene product has a homologous function and structure with the principal sigma factor of Escherichia coli was cloned and sequenced. The DNA region corresponding to one of the two hybridization signals found in P. aeruginosa DNA with a synthetic oligonucleotide probe (rpoD probe) was shown to be able to complement a temperature sensitive mutation of Escherichia coli rpoD gene. The amino acid sequence deduced from the nucleotide sequence of rpoDA showed an extensive homology with that of the principal sigma factor of E. coli throughout the entire region, which indicates that the two gene products have an essentially identical domain structure. A common basic structure observed among principal sigma factors of different eubacterial strains was proposed. RpoDA protein was identified in the extract of the cell carrying a plasmid clone with the rpoDA gene insert by Western blot analysis.     

Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp. strain PCC 7002.

The recA gene of Synechococcus sp. strain PCC 7002 was detected and cloned from a lambda gtwes genomic library by heterologous hybridization by using a gene-internal fragment of the Escherichia coli recA gene as the probe. The gene encodes a 38-kilodalton polypeptide which is antigenically related to the RecA protein of E. coli. The nucleotide sequence of a portion of the gene was determined. The translation of this region was 55% homologous to the E. coli protein; allowances for conservative amino acid replacements yield a homology value of about 74%. The cyanobacterial recA gene product was proficient in restoring homologous recombination and partial resistance to UV irradiation to recA mutants of E. coli. Heterologous hybridization experiments, in which the Synechococcus sp. strain PCC 7002 recA gene was used as the probe, indicate that a homologous gene is probably present in all cyanobacterial strains.     

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.     

Cloning, sequencing and overexpression of the gene for prokaryotic factor EF-P involved in peptide bond synthesis.

A soluble protein EF-P (elongation factor P) from Escherichia coli has been purified and shown to stimulate efficient translation and peptide-bond synthesis on native or reconstituted 70S ribosomes in vitro. Based on the partial amino acid sequence of EF-P, 18- and 24-nucleotide DNA probes were synthesized and used to screen lambda phage clones from the Kohara Gene Bank. The entire EF-P gene was detected on lambda clone #650 which contains sequences from the 94 minute region of the E.coli genome. Two DNA fragments, 3.0 and 0.78 kilobases in length encompassing the gene, were isolated and cloned into pUC18 and pUC19. Partially purified extracts from cells transformed with these plasmids overrepresented a protein which co-migrates with EF-P upon SDS polyacrylamide gel electrophoresis, and also exhibited increased EF-P mediated peptide-bond synthetic activity. Based on DNA sequence analysis of this gene, the EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,447. The sequence and chromosomal location of EF-P establishes it as a unique gene product.     

家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析

吡哆醛激酶（pyridoxal kinase,PLK, EC2.7.1.35）是维生素B₆关键代谢酶,其cDNA的克隆在昆虫类还未见报道。利用生物信息学原理和使用PCR方法,克隆出编码家蚕Bombyx mori吡哆醛激酶的cDNA (GenBank登录号DQ452397),体外原核表达成功,并对表达粗提产物进行了酶活检测。克隆到的cDNA含有一894 bp的完整可读框,编码一条分子量为33.1 kD,含298个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类吡哆醛激酶具有52.84%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但比哺乳动物和植物克隆到的吡哆醛激酶均少10多个氨基酸残基,几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换。依据家蚕基因组数据库信息和PLK的cDNA,家蚕PLK基因包含5个外显子和4个内含子,跨越10 kb DNA序列,所有外显子/内含子交接点都遵从gt/ag剪接规则,基因的5′端启动子调控区发现有TATA-box和CAAT-box保守基序。