Molecular cloning and nucleotide sequence analysis of the maltose-inducible porin gene of Aeromonas salmonicida

Abstract The gene for the Aeromonas salmonicida maltose-inducible porin (maltoporin) was cloned into phagemid pTZ18R in two restriction fragments, 0.6-kb Pst I/ Kpn I and 1.7-kb Sph I, of genomic DNA and their nucleotide sequences were determined. Open reading frames of 1329 and 1335 bp translated into sequences of 443 and 445 amino acids, with a 23 or 25 amino acid signal sequence and a 420 amino acid mature protein of molecular mass 46424 Da. Putative ribosome binding sites, AGGA and GGGAA, occurred 9 bp upstream of two possible ATG initiation codons. The A. salmonicida gene product showed a high degree of similarity with Escherichia coli LamB, and codon usage was very similar to that of another A. salmonicida outer membrane protein but markedly different from those of extracellular proteins.     

Effect of Vibrio cholerae non-O1 protease on lysozyme, lactoferrin and secretory immunoglobulin A

Abstract The effect of Vibrio cholerae non-O1 protease on host defense proteins (lysozyme, secretory immunoglobullin A and lactoferrin) was studied in relation to its virulence mechanism. The proteins treated with the protease were analysed by SDS-PAGE. There was no influence of the protease on lysozyme. The protease cleaved lactoferrin into two fragments of 50 kDa and 34 kDa. N-terminal amino acid sequencing of these fragments revealed that the cleavage site was near the hinge region, between serine 420 and serine 421. This cleavage could affect the transition from open to closed configuration which is involved in iron binding and release. The anti-bacterial activity of lactoferrin was not affected by protease treatment. Secretory immunoglobulin A yielded a 42-kDa protein as the cleavage product. The susceptibility of secretory immunoglobulin A to V. cholerae non-O1 protease suggests a mechanism by which bacteria might evade the effect of this immunoglobulin.     

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin)

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.     

Molecular cloning and functional expression of a fibrinolytic protease gene from the polychaeta, Periserrula leucophryna

Full-length cDNA encoding a fibrinolytic protease (PLFP) from the cDNA library of the polychaete, Periserrula leucophryna was cloned, sequenced and expressed in Escherichia coli. The entire cDNA of the PLFP clone was 921 bp (CDS: 41-837), including a coding nucleotide sequence of 798 bp, a 5′-untranslaed region of 40 bp, and a 3′-noncoding region of 83 bp. The ORF encoded a 265-amino acid polypeptide precursor consisting of a 36-residue signal sequence and a 229-residue mature polypeptide. The sequence alignment results of PLFP revealed sequence similarity with several fibrinolytic enzymes. Sequence analysis revealed a conserved catalytic triad of His78, Asp126 and Ser219 residues, suggesting that PLFP is a serine protease. Mature PLFP had an apparent molecular weight of approximately 25 kDa and was produced in inclusion bodies when expressed in E. coli. Substrate specificity results that recombinant PLFP was active towards Arg-X or Lys-X and did not hydrolyze substrates with nonpolar amino acids at the P1 site. Recombinant PLFP was strongly inhibited by typical serine protease inhibitors, further indicating that PLFP is a member of the serine protease family. PLFP was able to dissolve artificial plasminogenfree fibrin, and its fibrinolytic behavior was similar to that of plasmin. Taken these results together, PLFP is a new member of the fibrinolytic enzyme family with selective specificity on fibrin, and the availability of PLFP offers an attractive alternative approach for thrombolysis therapy because rPLFP is believed to have advantages over currently used plasminogen activators, that is, lower price and lower side effect.     

Nucleotide sequence and expression of nifHD from Frankia strain EuIKl, a symbiont of Elaeagnus umbellata

Nucleotide sequences of nif HD from Frankia strain EuIKl were determined and analysed. The 3.2-kb and 5.5-kb Bam HI fragments of a genomic clone were previously shown to contain nif HD-like sequences and to be contiguous, based on hybridization experiments and partial sequencing data. Sequence analysis of about 3.0 kb from these fragments revealed two open reading frames and beginning of a third in the same orientation, each of which showed high degree of sequence homology with nif H, nif D and nif K, respectively. The deduced amino acid sequence of the nif H ORF, consisting of 861 bp, showed sequence similarity of about 91% with those of other Frankia strains, whereas that of nif D ORF, consisting of 1458 bp, showed about 85% similarity. Intergenic sequence between nif H and nif D was 45 bp and between nif D and nif K was 61 bp. The 5'of nif H revealed putative Shine-Dalgarno sequences; however, a sequence resembling a typical nif -promoter or NifA binding site was not found. Northern hybridization of RNA from the nodules showed that nif HDK were transcribed into a polycistronic mRNA in the symbiont of Elaeagnus umbellata .     

Molecular cloning and characterization of a serine protease-like protein from silkworm (Bombyx mori)

TheBombyx mori (B. mori) serine protease-like protein (BmSp) coding region (946 bp, GenBank accession number of mRNA, DQ118520; protein, AAZ40503) was generated from two separate and overlapping cDNA fragments using sequence homology withTrichoplusia ni azurocidin in aBombyx EST database (Silkbase; http://www.ab.a.u-tokyo.ac.jp/silkbase/). The deduced amino acid sequence of BmSp, which encodes 303 amino acids, shows 44% amino acid identity toA. gambiae serine protease (CAA89967), 43% amino acid identity toSarcophagi peregrina 26-kDa protease, an antibacterial protein and 31% identity toB. mori serine protease-2 (BmSP-2), a potential antiviral protein. Typical features of the BmSp included the serine protease active site triad His / Asp / Ser, three pairs of cysteine residues for disulfide bridges, and three residues, Asp / Gly / Gly, that help to confer trypsin-like specificity to the enzymes. Based on the result of sequence comparison and characterization, our results suggest that the BmSp probably the new subfamily of trypsin-like serine protease. Using RT-PCR and enzyme digestion, the full encoding sequence for BmSp was cloned into theE. coli expression vector pGEX-5X-1. The fusion protein GST-BmSp was effectively expressed inE. coli BL21(DE3) pLysS as inclusion bodies, and a denaturation and refolding procedure were performed to obtain soluble GST-BmSp. The purified protein was tested for antibacterial activity against Gram-positive and Gram-negative bacteria, but it did not show antibacterial activity in the agar well diffusion assay and liquid growth inhibition assay.     

用改良消减杂交筛选类风湿性关节炎滑膜细胞中高表达基因

 为了研究类风湿性关节炎 (rheumatoid arthritis,RA)滑膜细胞 (fibroblast- like synovialcells,FLS)过度增殖和破坏软骨的分子机理 ,利用改良消减杂交法以骨性关节炎 (osteoarthritis,OA)病人滑膜细胞为对照 ,筛选 RA滑膜细胞中的高表达基因 .将得到的基因片段克隆入质粒载体 ,通过反向点杂交排除假阳性克隆后 ,将阳性克隆进行核酸序列分析 ,最后用 Northern杂交方法检测一些高表达基因在 RA和 OA病人滑膜细胞中的表达水平 .结果显示 ,共分离到 1 50个 RA高表达基因片段 ,其中长于 1 0 0 0 bp的片段占 8% (1 2 /1 50 ) ,长于 40 0 bp的片段占 36.7% (55/1 50 ) ,在大于 40 0 bp的片段中 ,假阳性率为 2 3.7% (1 3/55) .在测序的 1 8个片段中 ,已知基因有 1 2个 ,其中包括 IGF- 1结合蛋白 (IGFBP)特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B等 .新序列有 6个 ,其中两个序列分别与 Ring- box蛋白 1和 SON DNA结合蛋白同源 .对 IGFBP特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B基因的 Northern杂交分析显示 ,在 RA病人滑膜细胞中 ,这些基因的表达水平高于 OA病人滑膜细胞 .这些结果提示 ,这种改良消减杂交法是一种简便有效的分离差异表达基因的方法 ;IGF- 1结合蛋白特异性丝氨酸蛋白酶、层粘     

Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.     

Yeast thioredoxin genes

Based on the conserved protein sequence of thioredoxins from yeast and other organisms, two primers were synthesized for polymerase chain reaction of yeast genomic DNA. A 34-base pair (bp) sequence around the active site of yeast thioredoxin was obtained from the polymerase chain reaction product. This specific sequence was used as a probe in Southern blot analysis of total yeast genomic DNA digested with various restriction enzymes. Under conditions of high stringency, more than one DNA species hybridized with the probe, suggesting that more than one gene encodes yeast genomic library. Two Sau3A1 fragments, 825 and 2045 bp, respectively, from two different clones were cloned into pUC13. Sequence analysis of these fragments gave two different open reading frames without introns. The 825-bp Sau3A1 fragment encodes a 103-amino acid residue protein named thioredoxin I. The 2045-bp Sau3A1 fragment contains a sequence encoding thioredoxin II which has 102 amino acid residues. This is the first report of the cloning and sequencing of eukaryotic thioredoxin genes from any source. Both yeast thioredoxins contain a dithiol active site sequence, Cys-Gly-Pro-Cys. Thioredoxins I and II show 78% amino acid sequence identity. They display more amino acid sequence similarity with mammalian thioredoxin than with Escherichia coli and plant chloroplast thioredoxins.     

Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70 degrees C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes.     

Molecular analysis of one of multiple protease-encoding genes from the prototype virulent strain of Bacteroides nodosus

The aim of these studies was to examine the organization of the Bacteroides nodosus protease-encoding gene(s). The extracellular serine proteases (38 kDa) from the prototype virulent strain of B. nodosus were purified and used to raise a specific antiserum in rabbits. This antiserum was used in a colony immunoassay to screen a genomic DNA library constructed in Escherichia coli using BamHI-digested B. nodosus DNA and the plasmid pBR322. An E. coli clone expressing a 50-kDa immunoreactive polypeptide was identified. No protease activity was detected in the culture media, or in crude soluble and membrane fractions prepared from this clone. Restriction mapping and deletion analysis of the recombinant plasmid, pEKM2, was used to locate the coding region to a 1.4-kb EcoRI-BamHI fragment which was subsequently sequenced. A large open reading frame was found to extend through the BamHI site from a putative start codon just downstream from the EcoRI site, which indicated that the complete gene was not isolated. Southern blotting demonstrated that there were at least three B. nodosus BamHI fragments which were homologous to the 0.4-kb PstI-BamHI fragment of pEKM2. Based on these results the existence of multiple protease genes in B. nodosus was postulated.     

RNKP-1, a novel natural killer-associated serine protease gene cloned from RNK-16 cytotoxic lymphocytes

From an RNK-16 lambda-gt11 library, we have isolated and sequenced a novel cDNA rat NK cell protease 1 (RNKP-1) that has characteristics unique to serine proteases. The cDNA clone is 1102 bp and contains a complete open reading frame with an AUG start codon and a TAA stop codon. The open reading frame translates into a protein of 248 amino acids that has one glycosylation site. The characteristic N-terminal Ile-Ile-Gly-Gly and the His, Asp, and Ser amino acid residues that form the catalytic triad of serine proteases are present. The nucleotide and amino acid sequences have 87 and 80% identity, respectively, with the murine CTL-specific serine protease CCPI. However, there are extensive differences in the substrate binding regions of these proteases. Comparison of hydropathic profiles and amino acid sequences of other proteases indicate that RNKP-1 is distinct and belongs to the subfamily of serine proteases of bone marrow origin. Northern blot analysis of poly A+ RNA from rat splenocytes cultured with Con A showed 1000 and 1400 nucleotide mRNA are detected with RNKP-1 after 1 day of Con A-stimulation. The expression of the two mRNA bands continues through day 5 of culture with the lectin and may represent RNKP-1 mRNA plus related sequences due to cross-hybridization. RNKP-1 is also expressed in RNK-16 cells, but is not expressed in freshly isolated rat splenocytes, brain, lung, or lymph node tissues. The induction of RNKP-1 expression in the Con A-cultured spleen cells is accompanied by increases in both NK and lymphokine-activated killer lymphocyte activities. These data indicate that RNKP-1 is a unique serine protease that may be preferentially expressed in NK cells.     

Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence.

Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.     

Polymorphism and evolution of collagenolytic serine protease genes in crustaceans.

Two genomic DNA fragments encoding crustacean collagenolytic serine protease genes show coding fragments that span 1522-1526 base pairs and contain seven exons encoding the complete amino acid sequence of two enzymes, CHYA and CHYB. As in serine protease genes from other organisms, the region coding for the residues around the active site is split by two introns. Although the introns differ from those of other organisms in size and nucleotide sequence, their number and location are more or less the same as found in mammalian chymotrypsin or elastase genes that evolved lately, but different for trypsin genes. Meanwhile, the junction that occurs between the propeptide and the maturation site is only found in the shrimp genes. This is also the case for the junction located 13 amino acids after the active site aspartic acid in these genes. Between 40 and 50 copies of the genes are reported by Southern analysis. Seven different genes within ChyA Pv family present 0-6% base changes, whereas five different genes belonging to ChyB Pv family show changes of up to 27% in the short studied portion of exon 4. This last family presents a mosaic organization of the coding parts, which are also expressed in the hepatopancreas of the shrimp as the variant PVC5 cDNA.