Characterization, cloning and expression of the ferritin gene from the Korean polychaete, Periserrula leucophryna

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.     

cDNA cloning of a novel cysteine protease of Plasmodium falciparum

The cDNA for a novel Plasmodium cysteine protease (falcipain-2) has been isolated from a Plasmodium falciparum cDNA library. A 602 bp fragment was amplified from P. falciparum by PCR using degenerate oligonucleotide primers. The primers were designed based upon the amino acids flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases. This fragment was used to screen a P. falciparum cDNA library and isolated a 2.1 kb clone that encoded a novel cysteine protease. The sequence of the 2.1 kb clone predicted a 56 kDa protein containing a typical signal sequence, a prosequence and a 24.7 kDa mature protease with 37% identity to falcipain-1, a hemoglobin-degrading cysteine protease of P. falciparum. Northern blot analysis detected a 2.1 kb message in trophozoites. Taken together, we have isolated a novel cysteine protease of P. falciparum, which may play an important role at the late stages of the erythrocytic cycle of the parasite.     

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.     

Cloning and sequence analysis of Hemonchus contortus HC58cDNA.

The complete coding sequence of Hemonchus contortus HC58cDNA was generated by rapid amplification of cDNA ends and polymerase chain reaction using primers based on the 5' and 3' ends of the parasite mRNA, accession no. AF305964. The HC58cDNA gene was 851 bp long, with open reading frame of 717 bp, precursors to 239 amino acids coding for approximately 27 kDa protein. Analysis of amino acid sequence revealed conserved residues of cysteine, histidine, asparagine, occluding loop pattern, hemoglobinase motif and glutamine of the oxyanion hole characteristic of cathepsin B like proteases (CBL). Comparison of the predicted amino acid sequences showed the protein shared 33.5-58.7% identity to cathepsin B homologues in the papain clan CA family (family C1). Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the CBL, suggesting that HC58cDNA was a member of the papain family.     

Mouse cathepsin F: cDNA cloning, genomic organization and chromosomal assignment of the gene

A murine cysteine protease of the papain family was identified by dbEST-database search. A 1.87kb full-length cDNA encoding a predicted polypeptide of 462 amino acids was sequenced. Since the encoded polypeptide shows more than 80% sequence identity with human cathepsin F, it is most likely that this cDNA represents the murine homologue of cathepsin F, and it was therefore named accordingly. Murine cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 20 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 228 amino acids and the domain of the mature protease comprising 214 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the 'catalytic triad'. Genomic clones covering the murine cathepsin F gene were isolated. The mouse cathepsin F gene consists of 14 exons and 13 introns and spans 5.8kb. Murine cathepsin F was mapped to chromosome 19, a region with synteny homology to a region of human chromosome 11 to which human cathepsin F has been mapped previously. Northern blot analysis of RNA from multiple tissues revealed a ubiquitous expression of cathepsin F in mouse and man.     

Molecular cloning and expression analysis of novel wheat cysteine protease

A cDNA clone encoding a novel papain-like cysteine protease was isolated from wheat germ (Triticum aestivum). This cDNA encoded a 371-residue protein, designated WCP2, composed of signal peptide followed by a propeptide and a mature protease containing active site residues that are highly conserved among the papain family. The mature WCP2 protein (26 kDa) was detected in the quiescent embryo and its level of expression in the germinating embryo was greatly increased.     

Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein

A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.     

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.     

Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch

Many studies have been carried on peptides and genes encoding scorpion toxins from the venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as Na+, K+ and Cl- channel modulators. In this study, a novel calcium channel toxin-like gene BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch. First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5'RACE technique. The peptide deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms.     

Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure

We have isolated cDNA clones encoding boar acrosin, a serine protease participating in the initial stage of fertilization, from boar testis lambda gt11 cDNA libraries. Nucleotide sequencing of the overlapping clones indicates that the composite cDNA inserts contain 1,391 base pairs coding for a 5'-untranslated region, an open reading frame, a stop codon, a 3'-untranslated region, and a poly(A)+ tail. A polyadenylation signal, AATAAA, is located 33 bases upstream from the start of the poly(A)+ tail. The amino acid sequence deduced from the cDNAs shows that boar acrosin is initially synthesized as a prepro-protein with a 16-residue signal peptide at the NH2 terminus. This signal sequence is followed by a 399-residue sequence corresponding to the acrosin zymogen. COOH-terminal sequence analysis of boar sperm 55-kDa proacrosin and its processed forms indicates that the mature acrosin molecule contains 322 amino acid residues in two polypeptide chains, a 23-residue light chain and a 299-residue heavy chain, with a combined molecular mass of 35,735 Da, and that the 55-kDa proacrosin molecule has 14-, 18-, and 43-residue segments as COOH-terminal extensions that are removed during proacrosin maturation. The COOH-terminal 43-residue segment is rich in proline residues, including an unusual repeat of 23 consecutive prolines. The deduced amino acid sequence of boar acrosin shows a high degree of identity with major portions of other serine proteases, including the active site region and the location of cysteine residues. We conclude that boar acrosin is synthesized as a single-chain polypeptide with the regions corresponding to the light and heavy chains covalently connected by two disulfide bonds, and that the single-chain molecule is autoactivated by cleavage of the Arg23-Val24 bond after removal of the COOH-terminal 14-residue segment, resulting in the formation of the light and heavy chains. This two-chain molecule is then converted to the mature enzyme by removal of the COOH-terminal 18- and 43-residue segments.     

盐穗木甜菜碱醛脱氢酶基因（BADH）的克隆及其在盐胁迫下的表达分析

根据我们实验室已发表的植物甜菜碱醛脱氢酶基因（BADH）的同源保守区设计引物,通过RT—PCR扩增获得了由1503个核苷酸组成的盐穗木BADH基因开放阅读框,推测该基因编码500个氨基酸,分子量约为54．49kDa的多肽。推测的盐穗木BADH氨基酸序列中包含一段甜菜碱醛脱氢酶中高度保守的十肽序列（VTLELGGKSP）以及与酶功能有关的半胱氨酸残基（Cys）。序列比对结果显示,盐穗木BADH与盐地碱蓬、中亚滨藜、盐爪爪以及菠菜等的核苷酸序列同源性在81％以上,与水稻的同源性也达到68％。半定量RT—PCR分析结果表明,盐穗木BADH基因的表达受盐胁迫诱导,推测BADH可能与盐穗木具有较强的耐盐能力有关。     

Isolation of cDNA from Jacaratia mexicana encoding a mexicain-like cysteine protease gene

Cysteine proteases (CPs) from the C1 family, which are similar to papain, can be found in animals and plants, as well as some viruses and prokaryotes. These enzymes have diverse physiological functions and are thus very attractive for science and industry. Jacaratia mexicana, a member of the Caricaceae plant family, contains several CPs, the principal being mexicain, found to favorably compete against papain for many industrial applications due to its high stability and specific activity. In this study, leaves of J. mexicana were used to isolate a CP-coding gene, similar to those that code for mexicain and chymomexicain. By using rapid amplification of cDNA ends (RACE) as well as oligonucleotide design from papain-like conserved amino acids (aa), a sequence of 1404 bp consisting of a 5' terminal untranslated region (UTR) of 153 bp, a 3' terminal UTR of 131 bp, with a polyadenylation (poly(A)) signal sequence and a poly(A) tail, and an open reading frame (ORF) of 1046 bp, was obtained by overlapping three partial sequences. Two full-length cDNA sequences that encode for mexicain-like proteases were cloned from mRNA (JmCP4 and JmCP5). JmCP4 is predicted to have an ORF of 1044 bp, which codifies for polypeptides that have a 26 aa signal peptide region, a 108 aa propeptide region and a mature enzyme of 214 aa. A 969 bp fragment (JmCP5) encodes for a partial sequence of a CP gene, without the signal peptide region but with a full-length propeptide region. The sequence analysis showed that this protease presented a high similarity to other plant CPs from J. mexicana, Vasconcellea cundinamarcensis, Vasconcellea stipulata, and Carica papaya, among others, mainly at the conserved catalytic site. Obtaining the sequence of this CP gene from J. mexicana provides an alternative for production in a standard system and could be an initial step towards the commercialization of this enzyme.     

Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16h after injury and injection of bacteria.     

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge.     

Genetics and expression of two pectinesterase genes in Valencia orange

The genetics and expression of pectinesterase (PE) genes were examined in Valencia orange. Degenerate primers based on partial amino acid sequence of a 36 kDa PE protein isolated from juice vesicles were used to amplify a 350 bp DNA fragment from cDNA prepared from juice vesicle total RNA. Two groups of 350 bp PE clones with 66% sequence identity were isolated. A clone from each group was used to screen a Valencia orange genomic DNA λ library. Two different lambda clones that contained complete PE coding sequence (CsPME1 and CsPME3) and a third lambda clone that contained partial PE sequence (CsPME2) were characterized. The CsPME1 gene contained two exons (1063 and 689 bp) interrupted by a 1452 bp intron, whereas the CsPME3 gene had two exons (844 and 686 bp) interrupted by a 771 bp intron. CsPME1 shared significant sequence similarity with the partial clone CsPME2, including the entire cloned region of the first exon, a large region in the 5′ portion of the intron and the 3′ portion of the second exon, but the 3′ portion of the intron and the 5′ portion of the second exon were dissimilar. Southern blots suggested that Valencia orange has two genes within each PE group. Full-length cDNA clones that shared 99% sequence identity with CsPME1 and CsPME3 were isolated. Both groups of PE genes were differentially expressed in tissues of Valencia orange, and in addition CsPME3 appeared to be ethylene-regulated. The deduced proteins of PE cDNA clones CsPME1 (63.5 kDa) and CsPME3 (56.3 kDa) were considerably larger than the PE protein we isolated from Valencia orange juice vesicles and also other mature plant PE proteins. The estimated size of group I (2.2 kb) and group II (2.0 kb) PE mRNAs also predicted a larger protein than was isolated from juice vesicles. Alignment of the mature tomato and mung bean PE proteins, the most N-terminal sequence we obtained from polypeptides derived from the 36 kDa PE isolated from juice vesicles and the deduced amino acid sequences of plant PE cDNA clones suggest that a post-translational cleavage event separates the variable N-terminus from the more conserved C-terminal domain of the mature PE protein.