Cloning, sequencing and expression in Escherichia coli of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic lipase of Bacillus thermoleovorans ID-1 was cloned and sequenced. The lipase gene codes 416 amino acid residues and contains the conserved pentapeptide Ala-X-Ser-X-Gly as other Bacillus lipase genes. The optimum temperature of the lipase is 75 degrees C, which is higher than other known Bacillus lipases. For expression in Escherichia coli, the lipase gene was subcloned in pET-22b(+) vector with a strong T7 promoter. Lipase activity was approximately 1.4-fold greater than under the native promoter.     

Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in Escherichia coli

Summary A cyclcodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at –108 to –67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (M _r) and enzymatic properties as follows: M _r, 80 000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55°C; and stability below 45°C. The yields of the products from starch as the substrate were 25% for -and 5% for -cyclodextrin.The nucleotide and deduced amino acid sequence data reported in this paper have been deposited in the DDBJ, EMBL and Genbank Nucleotide Sequence Databases under the accession number D90243 Offprint requests to: T. Uozumi     

Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli

Bacillus subtilis strain B10 was isolated for degumming of ramie blast fibers, and a fragment of 642-bp was amplified from chromosomal DNA by using primers directed against the sequence of Bacillus subtilis xylanase gene given in GenBank. The positive clones were screened on the selected LB agar plates supplemented with xylan by Congo-red staining method. The recombinant plasmid from one positive clone was used for further analysis and DNA sequencing. The gene sequence is different from the reported xylanase gene sequence in sites of two base pairs. The recombinant plasmid was expressed in Escherichia coli, and xylanase activity was measured. The xylanase distribution in extracellular, intracellular and periplasmic fractions were about 22.4%, 28.0% and 49.6%, respectively. The xylanase had optimal activity at pH 6.0 and 50 degrees C.     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase.

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.     

Cloning, sequencing, and expression of porcine interleukin-18 in Escherichia coli

IL-18 is the new name of a novel cytokine that plays an important role in T(H1) response, primarily by its ability to induce IFN-gamma production in T cells and natural killer cells. The porcine IL-18 gene was isolated using RT-PCR from porcine alveolar macrophages. Sequence analysis of the porcine IL-18 gene has demonstrated an open reading frame of 579 base pairs encoding 192 amino acids precursor protein with a predicted molecular mass of 22 kDa. The porcine IL-18 gene shares 84% and 89% similarity to the human and canine equivalents, respectively, at the nucleotide level. The cloned IL-18 was expressed in Escherichia coli and its expression was confirmed by SDS-PAGE and Western blotting.     

Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin.

A Streptococcus faecalis genomic bank was obtained by partial digestion with MboI and cloning into the SalI restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus pumilus gene for ferulic acid decarboxylase.

The Bacillus pumilus gene encoding a ferulic acid decarboxylase (fdc) was identified and isolated by its ability to promote ferulic acid decarboxylation in Escherichia coli DH5 alpha. The DNA sequence of the fdc gene was determined, and the recombinant enzyme produced in E. coli was purified and characterized.     

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli

Summary A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage host-vector system was shown to direct the synthesis of a thermostable -amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000.The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused -lactamase--amylase protein with amylase activity.     

Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli.

The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity.     

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.     

Cloning and expression in Escherichia coli of apolyurethane-degrading enzyme from Pseudomonasfluorescens

A polyester polyurethane (PU)-degrading enzyme, PU esterase, derived from Pseudomonas fluorescens, a bacterium that utilizes polyester PU as the sole carbon source,was purified to homogeneity as indicated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. This enzyme was a soluble, extracellular protein with a molecular mass of 48 kDa and was inhibited by phenylmethylsulfonylfluoride (PMSF). A genomic library of Ps.fluorescens was constructed using the Escherichia coli bacteriophage l vector lZAPII. A recombinant phage exhibiting activity against Impranil DLN was isolated. The geneencoding the polyurethanase (PUase) protein was subcloned into a plasmid expression vectorpT7-6 and expressed in E. coli. Upon expression, the PUase was secreted by the host,displayed esterase activity which was inhibited by PMSF, and in vivo ³⁵S-methionine labeling of the gene product encoded by the open reading frame of the clone insertrevealed a single polypeptide with a molecular mass of 48 kDa.     

Cloning and expression of the Escherichia coli recA gene in Bacillus subtilis

By means of homopolymer dG-dC tailing, using PstI linearized pBR327 as vector, we constructed small plasmids containing the entire Escherichia coli recA gene. The 1.8-kb inserts were recloned in the Bacillus subtilis expression vector pPL608 in a B. subtilis recE4 strain. Analysis of plasmid-coded proteins showed expression of the E. coli recA gene both in minicells and whole cells of B. subtilis. Expression was under control of the bacteriophage SP02 promoter, which is part of pPL608. A recA-expressing plasmid completely abolished the transformation deficiency of the recE4 mutant as well as its sensitivity to mitomycin C (MC). The expressed recA gene also restored recombination in other B. subtilis strains lacking the recE gene product. These results indicate a high similarity between the functions of the E. coli RecA and B. subtilis RecE proteins.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

Cloning,sequencing, and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene

Bacillus sp. B-0618 produces both creatinase (Cre; creatine amidinohydrolase; EC 3.5.3.3) and sarcosine oxidase (Sox; EC 1.5.3.1) enzymes when grown in the presence of an inducer, choline chloride. A genomic library of Bacillus sp. B-0618, prepared in the plasmid vector pACYC184, was screened to obtain a gene (sox) encoding Sox by a convenient colorimetric assay. A plasmid, pOXI101, isolated from a sox-positive clone, contained a 14.2-kb insert of Bacillus DNA. The nucleotide sequence of a 1.7-kb segment containing the sox gene was determined, and it was found that an open reading frame encoding a protein consisting of 390 amino acids was located upstream from the cre structural gene cloned previously. When a 1.6-kb XhoI-BglII fragment of pOXI101 was inserted into the pUC118 vector and introduced into Escherichia coli, transformants cultured in the absence of the inducer produced Sox about 50-fold more than Bacillus sp. B-0618 cultured in the presence of the inducer. The Bacillus Sox had the -¹¹Gly-X-¹³Gly-X-X-¹⁶Gly- sequence motif that is highly conserved in flavoproteins. We created an FAD-free Sox by changing ¹³Gly to Asp in the motif of the parental Sox by oligodeoxynucleotide-directed mutagenesis. The mutant protein no longer expressed the Sox activity, even on the addition of FAD.     

Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli.

The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb Bg/II fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.     

Cloning, sequencing, and expression of cscA invertase from Escherichia coli B-62.

We have isolated a 2.5-kb DNA fragment from plasmid pST5R7 encoding a sucrose utilization system from Escherichia coli B-62 which confers a sucrose-fermenting phenotype to transformed E. coli K-12 strains. DNA-sequence determination revealed one full-length open reading frame 98% identical to cscA, the sucrose-hydrolase (invertase) gene of the csc regulon from E. coli EC3132. Functional characterization indicates that high-level expression and limited periplasmic release of invertase is responsible for the sucrose-fermenting capacity of transformed E. coli K-12 strains carrying cscA.     

Cloning,sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme

The bovine pancreatic (bp-) DNase I gene has been cloned from bp-cDNA and expressed in E. coli. A polynucleotide sequence of 1295 base pairs was deduced from clones of the cDNA. The sequence showed an open reading frame which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pET-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleotide coding for Met–Ala at the 5′-end. The plasmid was transformed into E. coli strain DH5α and the active recombinant bovine (rb-) DNase I was produced after induction of protein synthesis. From the induced culture medium, rb-DNase I was purified by chromatography on a Mono Q column. The purified rb-DNase I showed a molecular mass of 29 kDa and had the same specific activity as bp-DNase I. The NH₂-terminus of rb-DNase I was Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bp-DNase I, Asn was not glycosylated.     

枯草芽孢杆菌C-36内切葡聚糖酶基因的克隆及其在大肠杆菌中融合表达

以自行分离筛选出的天然枯草芽孢杆菌（Bacillus subtilis）C-36的染色体DNA为模板,PCR扩增得到含有内切葡聚糖酶基因的DNA片段,将其克隆到pMD-18T载体中,序列分析表明,克隆得到的DNA片段全长1602bp,编码一个含有499个氨基酸的多肽。与其他芽孢杆菌内切葡聚糖酶基因序列比对,其核苷酸同源率为90％～93％,其编码的氨基酸序列的同源性在90％～98％,已将此基因注册GenBank（DQ782954）。将含内切葡聚糖酶基因的重组克隆质粒进行亚克隆,用Kpn I和EcoR I双酶切后,与相同酶切的表达载体pET-32a相连接,并导入大肠杆菌BL21中表达。蛋白质电泳实验结果表明在6．47×10^4处有表达蛋白带。经测定表达蛋白比酶活力达99．02U／mL,为出发菌C-36（63．78U／mL）的1．55倍。     

Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis.

A genomic library of Bacillus lyticus was constructed in lambda GEM 11 vector and screened for the xylanase gene using Congo red plate assay. A 16-kb fragment containing the xylanase gene was obtained which was further subcloned using Mbo I partial digestion in an E. coli pUC 19 vector. A 1.3-kb sub-fragment was obtained which coded for a xylanase gene of Mr 23,650 Da. This fragment was sequenced and the homology was checked with known xylanases. The maximum homology was 97%, which was obtained with an endo xylanase gene from Bacillus species at the DNA level, while the translated sequence showed only one amino acid change from alanine to serine at position number 102. Expression was checked in E. coli, using the native promoter, and an extracellular activity of 5.25 U/mL was obtained. Cloning of the gene was done in Bacillus subtilis using a shuttle vector pHB 201, which resulted in increasing the basal level xylanase activity from 14.02 to 22.01 U/mL.