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PubMed2003

Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis.

The first amino acid residue from a proinsulin gene was fused in frame with the last amino acid residue of the aprE signal peptide sequence from Bacillus subtilis, using an overlapping PCR methodology. For expression of the fused DNA the aprE regulatory region (aprERR) was used. A six-protease-deficient strain of B. subtilis with the hpr2 and degU32 mutations was constructed for overproduction of the recombinant protein. The production of proinsulin was carried out in a mineral medium which facilitated the purification of proinsulin. Samples were taken during growth and analyzed by RIA and Western blot. Proinsulin was overproduced (1 mg ml(-1)) and 90% was secreted into the culture medium 1 h after stationary phase began.

Bacillus subtilis↗
PubMed2006

Combined amino acid mutations occurring in the envelope closely correlate with pathogenicity of EIAV.

The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system to study the attenuation mechanism and immunological control of lentivirus replication. Critical consensus mutations were identified between virulent Chinese EIAV strains and vaccine strains. Based on a full-length infectious clone of EIAV vaccine strain pLGFD3, two molecular clones, mFD5-4-7 and mFD7-2-11, were successfully constructed, in which 4 and 6 critical consensus mutations in the env gene of the vaccine strain were point-mutated to the wild-type sequence, respectively by an overlap PCR mutagenesis strategy. The infectivity, virulence, and pathogenesis of the constructed clones were investigated in vitro using a reverse transcriptase assay, an indirect immunofluorescence assay, observation of cytopathogenic effect, and virion observation as well as in vivo by inoculation of animals with the resulting infectious clones. The pathogenic symptoms in horses inoculated with mFD7-2-11 were more severe than those inoculated with mFD5-4-7, whereas no pathogenic symptoms were detected in animals inoculated with their parental clone pLGFD3 strain. The results indicate that the consensus mutation residues of the env region involved in this study play significant roles in the virulence and pathogenicity of EIAV. This will contribute to the elucidation of the attenuating and protective mechanisms of the Chinese EIAV vaccine.

Amino Acid Sequence↗
PubMed1996

Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fc epsilon receptor by a recombinant human autoantibody Fab converted to IgE.

Fc epsilon receptor (CD23)-mediated capture of IgE-antigen complexes by B cells provides a powerful antigen presenting system. Our goal was to develop a system using high affinity, human, organ-specific monoclonal autoantibodies for antigen capture by B cells. For this purpose, we converted a recombinant human autoantibody to TPO from a Fab (SP1.4) to an IgE molecule. Sera from all patients with autoimmune thyroid disease contain autoantibodies with the same epitope as SP1.4. The SP1.4 H and L chain V region genes were spliced by overlap PCR to a mammalian, non-immunoglobulin signal peptide and transferred to expression vectors for human IgG1 and kappa, respectively. After inserting the IgE constant region genes into the H chain vector, the kappa and IgE H chain vectors were expressed in SP2/0 cells. SP1.4-IgE retains its high affinity (Kd) for TPO (approximately 2 x 10(-10) M), recognizes the same epitope as Fab SP1.4 and, importantly binds to a different epitope than does Fab TR1.9. Binding of preformed complexes of SP1.4-IgE and biotinylated TPO to EB virus transformed B cells (EBVL) was weakly detectable by flow cytometry and was displaced by unlabeled TPO. SP1.4-IgE/125I-TPO complex binding to EBVL was much more clearly evident, was also inhibited by the addition of unlabeled TPO, and was greatly reduced by preincubation of the EBVL with anti-CD23. Further, autologous EBVL preincubated with SP1.4-IgE/TPO complexes stimulated proliferation of TPO-specific T cells. IgE autoantibody-mediated antigen focusing to B cells is unlikely to operate in vivo but is, instead, a powerful investigative tool. In conclusion, SP1.4-IgE is the first monoclonal human autoantibody to be developed for IgE-mediated antigen presentation to T cells by EBVL. Recombinant human autoantibodies converted to IgE, possibly in combinations if their epitopes permit simultaneous binding to the same molecule, provide a unique system to generate human T cell lines and clones specific for peptides naturally processed from internalized high affinity autoantibody/autoantigen complexes.

Antigen Presentation↗
PubMed1993

Nucleotide sequence analysis and epitope mapping of the merozoite surface protein 1 from Plasmodium chabaudi chabaudi AS.

The complete nucleotide sequence of the gene encoding the merozoite surface protein 1 (MSP-1) from the rodent malaria parasite Plasmodium chabaudi chabaudi AS has been determined by direct sequencing of overlapping PCR derived fragments. Comparison of the P. c. chabaudi AS nucleotide sequence with the previously published P. c. chabaudi IP-PC1 sequence indicates that although the MSP-1 gene of these two P. c. chabaudi strains is highly conserved, with sequence identity often approaching 100%, interspersed throughout the molecule are 5 regions of divergence. This is at variance with published data which suggested that the P. c. chabaudi AS and P. c. chabaudi IP-PC1 MSP-1 sequences are largely identical. Epitope mapping studies with a panel of anti-P. c. chabaudi AS MSP-1 monoclonal antibodies demonstrate that whilst most of these mAbs recognise epitopes at the N-terminus of the MSP-1 molecule, two mAbs, including one capable of inhibiting challenge infections in mice in an in vivo passive transfer assay, recognise epitopes which map to the C-terminus.

Amino Acid Sequence↗
PubMed2005

Sequences of cDNAs and expression of genes encoding chitin synthase and chitinase in the midgut of Spodoptera frugiperda.

The focus of this study was on the characterization and expression of genes encoding enzymes responsible for the synthesis and degradation of chitin, chitin synthase (SfCHSB) and chitinase (SfCHI), respectively, in the midgut of the fall armyworm, Spodoptera frugiperda. Sequences of cDNAs for SfCHSB and SfCHI were determined by amplification of overlapping PCR fragments and the expression patterns of these two genes were analyzed during insect development by RT-PCR. SfCHSB encodes a protein of 1523 amino acids containing several transmembrane segments, whereas SfCHI encodes a protein of 555 amino acids composed of a catalytic domain, a linker region and a chitin-binding domain. SfCHSB is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are expressed along the whole midgut. Chitin staining revealed that this polysaccharide is present in the peritrophic membrane (PM) only when SfCHSB is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHSB is responsible for PM chitin synthesis during the larval feeding stages and SfCHI carries out PM chitin degradation during larval-pupal molting, suggesting mutually exclusive temporal patterns of expression of these genes.

Amino Acid Sequence↗
PubMed2004

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.

Alternative Splicing↗
PubMed2006

Analysis of the near full length genomes of HIV-1 subtypes B, F and BF recombinant from a cohort of 14 patients in São Paulo, Brazil.

The human immune deficiency virus (HIV) exhibits strikingly tremendous amount of genetic variability. Such feature is critically important for the virus to adapt to environmental changes by escaping the host immune system and by escaping candidate vaccine. Therefore, understanding of such diversity is fundamental for the design of successful drugs or vaccine, which is urgently needed to bring the HIV/AIDS epidemic under control. In this study, we investigated the magnitude of diversity of the HIV-1 near full-length genomes from patients previously assigned as infected with non-recombinant HIV-1 subtypes B and F1 variants based on small portion of viral genome. HIV-1 proviral DNA was extracted from 14 samples previously classified in our laboratory as six subtypes B and eight subtypes F on the basis of small amplicon sequencing. Reamplifications of DNA from these samples were carried out by an overlapping PCR followed by direct sequencing. The data were phylogenetically inferred. Sequence analysis revealed that two out of six partially identified subtype B and six out of eight partially identified subtype F were in fact BF recombinants throughout their full genomes. Two pairs BF recombinants had identical genomic recombination structure and distinct from the Argentinean CRF 12_BF strains, probably represents a novel circulating recombinant forms in Brazil. Our data provided new genetic material of some of the HIV-1 subtypes currently circulating in the country and points to the widespread of BF recombinants which are expected to change the epidemic nature by approaching the level of subtype B in Brazil.

Adult↗
PubMed2004

Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL-37, in Escherichia coli.

The human cationic antimicrobial peptide hCAP-18/LL-37 is the unique cathelicidin identified in human to date. It has broad spectrum of antimicrobial activities and LPS-neutralizing activity and is involved in angiogenesis. Both purified and synthetic LL-37 or its derivatives were used in the study on LL-37. However, production of LL-37 in Escherichia coli has not been established. In this study, its precursor instead of the mature peptide was adopted for expression to avoid the lethal effect of recombinant LL-37 on host cells. A thrombin recognition site was introduced between the cathelin-like domain and LL-37 domain by overlap PCR to construct fragment encoding modified precursor (mhCAP-18) to facilitate the final release of the recombinant peptide. Then mhCAP-18 was fused in-frame to thioredoxin gene under the control of inducible T7 promoter to construct expression vector pET-mhCAP-18. The soluble form fusion protein was expressed in E. coli and purified by Chelating Sepharose column chromatography. Thrombin digestion of the fusion protein yielded recombinant GSLL-39, which was then purified by cation-exchange chromatography. Recombinant GSLL-39, which has two extra residues on its N-terminus when compared with its native counterpart, showed similar antimicrobial activities against both Gram-negative and Gram-positive bacteria.

Antimicrobial Cationic Peptides↗
PubMed2005

Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification.

Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.

Chromatography, Affinity↗
PubMed2005

Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference.

To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1-119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli (E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni-NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.

Base Sequence↗
PubMed2006

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH).

To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.

Animals↗
PubMed2005

Amino acid mutations of the infectious clone from Chinese EIAV attenuated vaccine resulted in reversion of virulence.

The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system by which attenuated mechanism and immunological control of lentivirus replication may be studied. We analyzed the critical consensus mutations that occurred during the viral passages in vitro and in vivo for vaccine's preparation. Based on the full-length infectious clone pLGFD3 (EIAV vaccine background) and according to mutations displayed during viral attenuation, we successfully constructed an infectious clones pLG5-3-l in which gag and env genes were point-mutated by overlap PCR mutagenesis strategy. pLG5-3-l was proved to have the ability of effective replication in vitro cells culture systems by Reverse Transcriptase Assay and virion observation under electron microscopy. Results of the in vivo experiments indicated that marked differences occurred between the mutated virus and their parental virus in clinical manifestation and plasma viral replication during 6-month observation period. In contrast to asymptom of animals infected with pLGFD3-V, the mutated virus (pLG5-3-l-V) developed typical clinical progression in the corresponding experimentally infected animals. The results of the distinct differences in clinical profiles and viral dynamics before and after mutation of EIAV infectious clone will help to understand the protective mechanism of Chinese EIAV vaccine and shed light on novel HIV vaccine design.

Amino Acid Substitution↗
PubMed1997

Neandertal DNA sequences and the origin of modern humans.

DNA was extracted from the Neandertal-type specimen found in 1856 in western Germany. By sequencing clones from short overlapping PCR products, a hitherto unknown mitochondrial (mt) DNA sequence was determined. Multiple controls indicate that this sequence is endogenous to the fossil. Sequence comparisons with human mtDNA sequences, as well as phylogenetic analyses, show that the Neandertal sequence falls outside the variation of modern humans. Furthermore, the age of the common ancestor of the Neandertal and modern human mtDNAs is estimated to be four times greater than that of the common ancestor of human mtDNAs. This suggests that Neandertals went extinct without contributing mtDNA to modern humans.

Animals↗
PubMed2002

Voltage-gated Ca2+ channel Ca(V)1.3 subunit expressed in the hair cell epithelium of the sacculus of the trout Oncorhynchus mykiss: cloning and comparison across vertebrate classes.

Full-length sequence (>6.5 kb) has been determined for the Ca(V)1.3 pore-forming subunit of the voltage-gated Ca(2+) channel from the saccular hair cells of the rainbow trout (Oncorhynchus mykiss). Primary structure was obtained from overlapping PCR and cloned fragments, amplified by primers based on teleost, avian, and mammalian sources. Trout saccular Ca(V)1.3 was localized to hair cells, as evidenced by its isolation from an epithelial layer in which the hair cell is the only intact cell type. The predicted amino acid sequence of the trout hair cell Ca(V)1.3 is approximately 70% identical to the sequences of avian and mammalian Ca(V)1.3 subunits and shows L-type characteristics. The trout hair cell Ca(V)1.3 expresses a 26-aa insert in the I-II cytoplasmic loop (exon 9a) and a 10-aa insert in the IVS2-IVS3 cytoplasmic loop (exon 30a), neither of which is appreciably represented in trout brain. The exon 9a insert also occurs in hair cell organs of chick and rat, and appears as an exon in human genomic Ca(V)1.3 sequence (but not in the Ca(V)1.3 coding sequence expressed in human brain or pancreas). The exon 30a insert, although expressed in hair cells of chick as well as trout, does not appear in comparable rat or human tissues. Further, the IIIS2 region shows a splice choice (exon 22a) that is associated with the hair cell organs of trout, chick, and rat, but is not found in human genomic sequence. The elucidation of the primary structure of the voltage-gated Ca(2+) channel Ca(V)1.3 subunit from hair cells of the teleost, representing the lowest of the vertebrate classes, suggests a generality of sensory mechanism for Ca(V)1.3 across hair cell systems. In particular, the exon 9a insert of this channel appears to be the molecular feature most consistently associated with hair cells from fish to mammal, consonant with the hypothesis that the latter region may be a signature for the hair cell.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species.

The femA gene encodes a protein precursor which plays a role in peptidoglycan biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance. A femA homologous gene was recently characterized in S. epidermidis, entailing the possibility of femA phylogenetic conservation in staphylococcal species. Accordingly, we assessed the presence of femA homologous genes in S. hominis and S. saprophyticus. Strategy for identification relied upon alignment of S. aureus and D. epidermidis femA sequences and upon identification of potentially conserved regions. Amplifications of portions of the femA genes were performed under permissive annealing conditions, by using several sets of primers designed to match the consensus regions. DNA sequencing of overlapping PCR fragments led to the characterization of the entire femA genes of S. hominis and S. saprophyticus, and provided more precise information on the femA start codon for all five species. The genomic organization of all these femA genes appeared highly conserved, with alternance of homologous and variable regions. On this basis, a consensus sequence of the femA gene was defined and interspecies variations were exploited to design strategies for staphylococci species-specific identification, including multiplex PCR amplification and a reverse hybridization assay.

Amino Acid Sequence↗
PubMed2003

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.

Alternative Splicing↗
PubMed2000

Cloning and expression of the bovine 11beta-hydroxysteroid dehydrogenase type-2.

The bovine 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11beta-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11beta-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5'-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3'-untranslated region of 530 bp. Bovine 11beta-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11beta-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11beta-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD(+) and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11beta-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2002

Expression, purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli.

Minimized proteins have long been used to elicit immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. Here we describe molecular cloning and production of a minimized chicken riboflavin carrier protein (mini-RCP) sequence that harbours all the four neutralizing epitopes but lacks the sequences that otherwise elicit undesirable antibodies. The gene encoding mini-RCP is engineered by contiguous alignment of nucleotide sequences coding for selected epitopes of chicken RCP separated by leucyl alanine residues. The gene has been constructed from eight oligonucleotides by employing overlapping PCR strategy and expressed in Escherichia coli, using the T7 promoter system. The recombinant protein could be purified to homogeneity by a single step Ni2+ affinity chromatography. Western blot experiments using epitope specific antisera confirm that the corresponding linear amino acid sequences are available for immunorecognition in the engineered protein. This methodology enables continuous production and purification in bulk amounts of the minimized RCP as a source of candidate immunocontraceptive vaccine in mammals.

Amino Acid Sequence↗