Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambdamax, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays.     

Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes

The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme.     

Regulation and secretion of an extracellular esterase from Streptomyces scabies.

Production of a heat-stable, extracellular esterase by Streptomyces scabies is regulated by zinc ions. The esterase-encoding gene (est) from S. scabies was cloned and expressed in Streptomyces lividans. In S. lividans, expression of the est gene is also regulated by Zn2+, and the esterase is efficiently secreted in this organism. The sequence of the est gene suggests that a 39-amino acid signal peptide is removed during secretion of this protein. Deletion analysis has indicated that the hydrophobic domain of the signal peptide is required for secretion. Gel retardation assays and DNaseI footprinting using an S-30 protein extract from S. scabies have previously identified a specific 23-bp protein-binding site upstream from the est coding sequence. Deletion of this protein-binding sequence significantly decreased expression of the est gene.     

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.     

Mouse preproacrosin: cDNA sequence, primary structure and postmeiotic expression in spermatogenesis

The primary structure of mouse preproacrosin was deduced by nucleotide sequencing of cDNA clones isolated from a mouse testis cDNA library. The largest cDNA, with 1373 bp, consists of a 11-bp 5'untranslated sequence, a 1254-bp open reading frame terminated by a TGA triplet and a 105-bp 3' untranslated end, including one potential polyadenylation signal. The NH2-terminus of the polypeptide contains a hydrophobic 15-amino acid signal peptide. This cleavable signal sequence is followed by 403 amino acids, representing the acrosin light and the heavy chain of 23 and 380 amino acid residues, respectively. The proteolytic active site segments His, Asp and Ser are part of the heavy chain, as well as a proline-rich COOH-terminus, which is not present in any other serine proteinase studied so far. Furthermore the postmeiotic expression of the preproacrosin gene during mouse spermatogenesis was studied.     

Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λ_max = 480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2–3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis.     

Exons of the human pancreatic polypeptide gene define functional domains of the precursor

Pancreatic polypeptide is a 36-amino acid peptide which inhibits pancreatic exocrine function. We have previously determined from the nucleotide sequence of a cDNA that pancreatic polypeptide is derived from a 95-amino acid precursor, prepropancreatic polypeptide. Pulse-chase studies have suggested that the precursor is cleaved to produce three peptides: pancreatic polypeptide, an icosapeptide, and a smaller peptide. In the present study, we have used the cloned cDNA as a hybridization probe to isolate the pancreatic polypeptide gene from a human bacteriophage genomic library. The nucleotide sequence of 2.8 kilobases of DNA representing the entire human pancreatic polypeptide gene was determined. The gene contains four exons and three introns. Exon 1 encodes the 5'-untranslated region of the mRNA, exon 2 encodes the signal sequence and the sequence of pancreatic polypeptide, exon 3 encodes the icosapeptide, and exon 4 encodes a carboxyl-terminal heptapeptide and the 3'-untranslated region of the mRNA. By Southern blot analysis, the gene detected in a pancreatic polypeptide-producing islet cell tumor was indistinguishable from that in normal human leukocytes. The structure of the human pancreatic polypeptide gene is consistent with the hypothesis that prepropancreatic polypeptide generates three distinct peptides, each encoded by a separate exon. Increased expression of pancreatic polypeptide in the islet cell tumor does not appear to be correlated with major alterations in pancreatic polypeptide gene structure.     

Cloning of the novel putative apoptosis-related gene of Spirometra erinacei (Order Pseudophyllidae)

We postulated that apolysis was processed in accordance with apoptotic changes occurring in a cestode, Spirometra erinacei (Pseudophyllidea). We cloned the novel putative apoptosis-associated gene from S. erinacei via screening of a S. erinacei cDNA library with a ced-3 gene (activator of apoptosis) probe from Caenorhabditis elegans. We identified a 261-bp cDNA sequence, which encodes for an 86-amino acid protein. The cloned gene expression was observed in the neck and gravid proglottids via Northern blotting, using cloned cDNA inserts as probes, but the clone was not expressed in any of other tissues. We suggest that this gene may be involved in the apolysis of S. erinacei during normal tissue development and differentiation in cestode parasites.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca

A secreted luciferase from the marine ostracod, Vargula hilgendorfii, is a useful tool for gene expression assays in living mammalian cells. We have cloned the cDNA of a new secreted luciferase from the ostracod Cypridina noctiluca, which inhabits the coast of Japan. C. noctiluca luciferase consists of 553 amino acid residues with a molecular mass of 61,415 Da, as deduced from the nucleotide sequence. The homologies of nucleotide and amino acid sequences with V. hilgendorfii luciferase are 79.2% and 83.1%, respectively. C. noctiluca luciferase can expressed in and secreted from cultured mammalian cells. The characteristic properties of expressed C. noctiluca luciferase are similar to those of V. hilgendorfii luciferase. However, the activity of C. noctiluca luciferase in culture medium is much higher than that of V. hilgendorfii luciferase, suggesting that C. noctiluca luciferase is a highly potent reporter enzyme for real-time and continuous monitoring of gene expression in living cells.     

Molecular Cloning and Expression of Proteinaceous α-Amylase Inhibitor Gene from Streptomyces nitrosporeus in Escherichia coli

The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.     

General Information

A novel cDNA clone encoding a COR413-like gene was isolated by suppression subtraction hybridization and cDNA library screening from sea-island cotton (Gossypium barbadense). This gene (designated as GbCOR413, Accession number: AY761065) has a total length of 893 bp with an open reading frame of 600 bp, encoding a predicated polypeptide of 200 amino acids with a molecular weight of 22.74 kDa and a predicated pI of 9.2. Bioinformatics analyses revealed that this gene belonged to a novel stress-regulated multi-spanning transmembrane protein family without signal peptide. By means of semi-quantities RT-PCR analysis, the expression of GbCOR413 under short-term cold treatment at 4°C, water submergence and abscic acid treatment was investigated. Our studies suggested that the cloned gene was a new member of COR gene family which was slowly responsive to cold stress in cotton. Jin Wang and Kai-Jing Zuo are co-first authors of this paper.     

Human Gastric Inhibitory Polypeptide Receptor: Cloning of the Gene (GIPR) and cDNA

Gastric inhibitory polypeptide (GIP), which is released from the gastrointestinal tract, stimulates insulin secretion from pancreatic β cells and plays a crucial role in the regulation of insulin secretion during the postprandial phase. We have isolated the human gene (GIPR) and cDNA encoding the GIP receptor by a combination of the conventional screening and polymerase chain reaction procedures. Human GIP receptor cDNA encodes a protein of 466 amino acids that is 81.5 and 81.2% identical to the previously cloned hamster and rat GIP receptor, respectively. Hydropathic analysis shows the presence of a signal peptide and seven potential transmembrane domains, a feature characteristic of the VIP/glucagon/secretin receptor family of G protein-coupled receptors. The human GIPR gene is about 13.8 kb long, consists of 14 exons, and carries 17Alurepeats.     

Peptide YY. Structure of the precursor and expression in exocrine pancreas

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.     

The conformation of mature human alpha-amylase conditions its secretion from yeast

The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.     

Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631

We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.     

Cloning and expression of a cDNA encoding the laccase from Schizophyllum commune

We cloned and analyzed the nucleotide sequence of a cDNA that encodes polyphenol oxidase (laccase) from the white-rot basidiomycete Schizophyllum commune. The nucleotide sequence of the full-length cDNA contained a 1554-base open reading frame that encoded a polypeptide of 518 amino acid residues, including a putative signal peptide of 16 residues. It contained four highly similar regions that are conserved in the deduced amino acid sequences of other laccases, including the region thought to be involved in copper binding. Aspergillus sojae strain 1860 (which has low protease levels) was transformed with the plasmid lacAL/pTPT, which contained the laccase gene under the control of the tannase promoter from Aspergillus oryzae. Laccase was secreted into the medium when transformants A1 and A2 were cultured in tannic acid-containing medium.     

Secretion of firefly luciferase in E.coli

Summary A DNA segment carrying the full-length, intronless firefly luciferase gene was inserted into the high expression secretion vector, pIN-III -ompA. Upon induction of gene expression, luciferase activity was detected in extracts prepared from periplasmic fractions. The results indicated that the OmpA signal peptide was able to direct secretion of firefly luciferase across the cytoplasmic membrane. This has important implications for using this luciferase as a reporter in studying protein export and targeting.     

Structure and activity of human pancreasin,a novel tryptic serine peptidase expressed primarily by the pancreas

In a search for genes encoding the serine peptidases prostasin and testisin, which are expressed mainly in prostate and testis, respectively, we identified a related, novel gene. Sequencing of cDNA allowed us to deduce the full amino acid sequence of the human gene product, which we term "pancreasin" because it is transcribed strongly in the pancreas. The idiosyncratic 6-exon organization of the gene is shared by a small group of tryptic proteases, including prostasin, testisin, and gamma-tryptase. Like the other genes, the pancreasin gene resides on chromosome 16p. Pancreasin cDNA predicts a 290-residue, N-glycosylated, serine peptidase with a typical signal peptide, a 12-residue activation peptide cleaved by tryptic hydrolysis, and a 256-amino acid catalytic domain. Unlike prostasin and other close relatives, human pancreasin and a nearly identical chimpanzee homologue lack a carboxyl-terminal membrane anchor, although this is present in 328-residue mouse pancreasin, the cDNA of which we also cloned and sequenced. In marked contrast to prostasin, which is 43% identical in the catalytic domain, human pancreasin is transcribed strongly in pancreas (and in the pancreatic ductal adenocarcinoma line, HPAC) but weakly or not at all in kidney and prostate. Antibodies raised against pancreasin detect cytoplasmic expression in HPAC cells. Recombinant, epitope-tagged pancreasin expressed in Chinese hamster ovary cells is glycosylated and secreted as an active tryptic peptidase. Pancreasin's preferences for hydrolysis of extended peptide substrates feature a strong preference for P1 Arg and differ from those of trypsin. Pancreasin is inhibited by benzamidine and leupeptin but resists several classic inhibitors of trypsin. Thus, pancreasin is a secreted, tryptic serine protease of the pancreas with novel physical and enzymatic properties. These studies provide a rationale for exploring the natural targets and roles of this enzyme.