Recombinant P1 Research Articles

Protective immune response against foot-and-mouth disease virus challenge in guinea pigs vaccinated with recombinant P1 polyprotein expressed in Pichia pastoris

Vaccination of the susceptible livestock with potent, safe and cost effective vaccine is the primary requirement to control foot-and-mouth disease (FMD) in an endemic country. In this study, an alternative approach was used in which structural protein genes of all the four serotypes of FMDV (O, Asia 1, A22 and C) were expressed separately in methylotrophic yeast Pichia pastoris. The recombinant polyproteins (P1) were characterized by SDS-PAGE and in Western Blot analysis. Partially purified protein was used for immunization in guinea pigs with different adjuvant formulations and immune response studied. Ninety micrograms of the recombinant protein per monovalent dose was used for immunization. A single injection of a monovalent or polyvalent vaccine was given to guinea pigs with various adjuvant combinations viz., Monovalent recombinant protein either adjuvanted with Montanide-ISA50V or Indigenous oil, Monovalent recombinant protein mixed with 1/10th dose of inactivated oil-adjuvanted virus vaccine and Polyvalent recombinant protein with Montanide ISA50V. FMDV specific humoral immune response was observed at about 28th day post vaccination. The immune response as assessed by indirect ELISA and Serum neutralization test titres was found to be 320-640 and 16-32, respectively. When challenged with virulent homologous type 'O' virus, the guinea pigs showed protective C index of 2.01,1.81, 2.56 and 2.48, respectively, with above said adjuvant combinations. The study has shown that yeast-expressed FMDV P1 polyprotein in a single dose could elicit a protective immune response in guinea pigs, and this could be a possible future vaccine candidate in homologous host.

Autoantibodies and Autoantigens in the Urine of SLE Patients

Autoantibodies against RNA polymerase I (RNAPI), DNA, La and ribosomal P proteins were detected in the urine of systemic lupus erythematosus (SLE) patients, many with normal protein excretion rates. In a number of cases, the antibodies were detectable in the urine but not the serum sample of the same patient. The presence and relative concentrations of the urinary autoantibodies correlated with disease activity. RNAPI antigens were detected in the urine of SLE patients by radioimmunoassay and immunoblotting using rabbit antisera prepared against the purified holoenzyme. Immunoaffinity purification of the rabbit anti-RNAPI with SLE urine proteins resulted in antibodies directed primarily against the largest RNAPI subunit (S1; 194 kDa). Antibodies prepared against recombinant fusion proteins representing the DNA binding regions of human RNAPI(S1) reacted with a 35 kDa SLE urinary protein, a putative fragment of RNAPI(S1). Ribosomal protein P0 was detected in SLE patients' urine by immunoblotting, using rabbit antiserum prepared against recombinant human P1 fusion protein. The relative quantities of urinary P0 correlated with disease status. Analysis of urinary autoantibodies and corresponding antigens in conjunction with analysis of serum autoantibodies may be of value for the purpose of monitoring disease activity.

Protective immune response of the capsid precursor polypeptide (P1) of foot and mouth disease virus type ‘O’ produced in Pichia pastoris

Foot and mouth disease virus (FMDV) is the aetiological agent of a highly contagious vesicular disease of cloven-hooved animals. The gene coding for the capsid polyprotein (P1) of FMDV from serotype ‘O’ vaccine strain (O75Madras) was cloned and expressed in yeast Pichia pastoris. The expressed P1 protein was characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analysis. Immunisation of Guinea pigs with recombinant P1 induced FMDV type O specific immune response. The humoral response to vaccine was measured by indirect ELISA and a serum neutralisation test (SNT). The Guinea pig sera showed high titres both in ELISA and SNT. Upon challenge with virulent Guinea pig adapted homologous type ‘O’ virus, the animals showed a protective index of 2.52. This study shows that the yeast expressed FMDV P1 could be a safe vaccine in non-endemic countries and a cost-effective vaccine in endemic countries. This is the first report on the production of FMDV structural proteins in yeast and their application as a vaccine.

The end of human DNA maps? Little P. Nat Genetics 2001;27:229–230.

The February 15 and 16, 2001 issues of Nature and Science, respectively, published maps and sequences of the human genome. This unprecedented accomplishment achieved worldwide attention. The issues of the journals contained the maps/sequences and accompanying articles. The maps/sequences were also published as charts and CDs that accompanied the issues. The Nature consortium was publicly funded in part by the Human Genome Project, and Celera privately funded the Science group. The Nature consortium broke up the DNA in fragments of approximately 150,000 base pairs, using recombinant bacterial artificial chromosomes or phage P1 artificial chromosomes (BACs or PACs, respectively). In essence, the entire human DNA is represented as a series of overlapping 150,000-bp fragments. It is a “map” of overlapping DNAs that is then used by the DNA sequencers who select individual clones (the “clone-by-clone” strategy) for sequencing, with an ultimate goal of filling in the framework of the genome with the DNA sequence. By contrast, the Science group directly analyzed all 3,000 million bases of human DNA, which has been broken up randomly into small fragments of a few thousands of base pairs (the “read” capacity of automated DNA analyzers is not more than 500–1,000 bp). Massive computational efforts were then used to read these short DNA sequences and determine how they overlap. The maps of clones have great usefulness in their own right. The maps/sequences have indicated that any two unrelated individuals differ by about 1 base every 1,000 bases, and the average gene contains 12 variations that are common within populations. Editor’s note: In this issue of the American Journal of Ophthalmology, an abstract of the Nature consortium’s physical map of the human genome is provided along with abstracts of accompanying Nature articles that show how physical maps of various human chromosomes are constructed. Also, an abstract of the Science group’s sequence of the human genome with abstracts of accompanying Science articles interpreting some of the identified genes is included. These maps and sequences of the human genome represent perhaps the most important single scientific accomplishment of this generation.—Hans E. Grossniklaus

Monoclonal antibodies against human ribosomal P proteins penetrate into living cells and cause apoptosis of Jurkat T cells in culture.

This study was designed to determine the role of autoantibodies to the ribosomal P protein (anti-P Abs) in the pathogenesis of systemic lupus erythematosus (SLE) using monoclonal anti-P antibodies (anti-P mAbs). Anti-P mAbs were prepared by a standard hybridoma procedure using recombinant human P1 and P2 proteins as immunogens. We studied the reactivities of these mAbs to P proteins, their binding and penetration capabilities in different cell lines and their apoptotic effects on Jurkat T cells. In addition to recognizing human P0, P1 and P2 proteins, the anti-P mAb 9B6-4 bound to 20-40% and penetrated 50-90% of astrocytes, Jurkat T cells and lung cancer cells via the P0 surface protein. Treatment with the mAb 9B6-4 also caused increases in the percentages of Jurkat T cells in the sub-G1 phase of the cell cycle (14.8%) and undergoing apoptosis (21.3%). Anti-P autoantibodies may play a role in the pathogenesis of lymphopenia or lymphocyte dysfunction in SLE.

Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or beta(2)-ARS:

Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus influenzae: an unambiguous, multifaceted approach.

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (</=95% similarity) were identified among 42 strains. When chinchillas were immunized with recombinant P1 (rP1) obtained from one of these isolates (BCH-3), all animals developed antibodies specific for rP1. Immunized animals were protected against disease when challenged with BCH-3, but not with an ompP1 mutant of BCH-3 or a strain (BCH-2) possessing a heterologous P1 (91% identity). We conclude that (i) while P1 induces protection against NTHI-mediated otitis media, development of a polyvalent vaccine reflecting the variability of P1 would be necessary to construct an efficacious vaccine and (ii) use of a phylogenically characterized collection of representative isolates in concert with gene sequencing, cloning, gene inactivation, and animal testing offers an efficient, rational, and rigorous strategy for evaluating the potential problems associated with variability of vaccine targets and specificity of related immune responses.

Effect of sequence variation in meningococcal PorA outer membrane protein on the effectiveness of a hexavalent PorA outer membrane vesicle vaccine

Though meningococcal serogroup C conjugate vaccines have been introduced into the UK infant immunisation schedule, there is currently no vaccine solution for serogroup B disease. PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate. A hexavalent PorA outer membrane vesicle (OMV) vaccine has been evaluated in phase I and II trials with promising results. This vaccine contains six different PorA OMPs each representing a different serosubtype. However, considerable sequence variation occurs in the variable regions (VRs) encoding these serosubtypes. By using recombinant P1.5,10 PorA variants we have demonstrated that the killing of this particular serosubtype combination was due mainly to the induction of antibody to the VR2 (P1.10) epitope region, and that after three or four doses of vaccine there was a significant reduction in the killing of variants P1.10a (three doses, p<0.0001; four doses, p=0.003) and P1.10f (three doses, p<0.0001; four doses, p=0.002), as compared to responses to the P1.10 strain, when the P1.10 serosubtype was used as the immunogen. Since large numbers of serosubtype variants are known to exist, this finding may have implications for the use of PorA as a meningococcal serogroup B vaccine.

RNA-binding activities of cocksfoot mottle sobemovirus proteins

Cocksfoot mottle virus (CfMV) has a positive-sense ssRNA genome containing four open reading frames (ORFs). ORF1 encoded protein (P1) is the putative movement protein; the product of ORF2a (P2a) contains VPg and the motifs characteristic of serine proteases. P2b, encoded by ORF2b, is the putative RNA-dependent RNA polymerase. P3, the coat protein, is encoded by ORF3. CfMV P1, P2a, P2b, and P3, containing a six histidine tag at the amino terminus, were expressed in Escherichia coli, purified and their RNA-binding activities were analysed. The northwestern blot assay showed that His-tagged P1, P2a, P2b, and P3 were able to interact with ssRNA transcripts in a sequence-nonspecific manner. The filter-binding assay confirmed the ssRNA-binding capacity of recombinant P1, P2a, and P3. The RNA-binding activities of His-tagged P3 and native coat protein were similar. P1 and P2a binding to ssRNA decreased markedly by increasing NaCl concentrations. In contrast, P3 had the RNA-binding optimum at 100–200 mM NaCl. We discuss the possible amino acid motifs involved in the RNA-binding of CfMV proteins.

Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus

Swine vesicular disease virus (SVDV) is the aetiological agent of a highly contagious viral disease of pigs, whose symptoms are indistinguishable from those caused by foot-and-mouth disease virus (FMDV). The gene coding for the capsid protein precursor of SVDV (P1) from a recent spanish isolate (SPA/1/'93) was cloned and expressed in bacteria, and the antigenicity and immunogenicity of the recombinant product were evaluated. The recombinant P1 was recognised by antibodies against SVDV induced in pigs infected experimentally with different SVDV strains. Immunisation of swine with recombinant P1-induced SVDV-specific cellular and humoral immune responses. The implications of these results in SVD diagnostic as well as in vaccine development are discussed.

Detection of potato virus Y P1 protein in infected cells and analysis of its cleavage site.

The P1 protein is liberated from the N-terminal region of the potyviral polyprotein by cleavage depending on its own autoproteolytic activity. Existence of the 32-kDa P1 protein in tobacco plants infected with potato virus Y ordinary strain (PVY-O) was detected by an antiserum against a recombinant PVY-O P1 protein. In vivo analysis using tobacco protoplasts confirmed that the Phe284-Ser285 was the cleavage site separating the P1 protein from the PVY-O polyprotein. Phe284 was indispensable for proteolysis and Ser285 was needed for optimal cleavage susceptibility.

The transcriptional regulatory strategy of the rat tissue kallikrein gene family

We have analysed the transcriptional regulatory strategy of the rat tissue kallikrein gene family, a strategy that provides universal submandibular gland expression of all family members coupled with otherwise disparate expression of individual members in a wide variety of organs. To test whether a locus control region (LCR) or individual gene enhancers control the family, the expression patterns of rat kallikrein genes were examined in transgenic mice bearing a series of rat genomic fragments spanning the kallikrein locus. Each fragment, present in recombinant P1 phage clones, contained two or three linked members of the rat family. Rat (r) genes KLK1, KLK2, KLK3, KLK7, KLK8, KLK9 and KLK10 on four different P1 clones were all correctly expressed at high levels in the submandibular glands of transgenic mice and in general showed the correct extra-salivary patterns characteristic of each family member. Moreover, when the neighbouring family members rKLK1 and rKLK3 were separated on non-overlapping fragments and tested in mice, each transgene was expressed correctly in the submandibular gland and in other organs characteristic of that gene. Thus the family locus is not controlled by an LCR; rather each gene appears to have an associated transcriptional enhancer that specifies high submandibular expression and contributes to the additional organ specificity of the family member.

Localization and intron usage analysis of the human CPT1B gene for muscle type carnitine palmitoyltransferase I

We isolated and sequenced cDNA and genomic DNA fragments of the human CPT1B gene, encoding muscle type carnitine palmitoyltransferase I. A recombinant P1 phage containing CPT1B was mapped to chromosome 22qter by fluorescent in situ hybridization. This finding supports the concept that `liver type' and `muscle type' isoforms of CPT I are encoded by different loci at separate chromosomal positions. Analysis of CPT1B cDNA sequences revealed the presence of an untranslated 5′ exon and differential processing of introns 13 and 19. The alternative splicing of intron 13 causes an in-frame deletion leading to a 10 amino acid residues smaller protein. Using different splice acceptor sites, intron 19 is spliced in the majority of cases, but 4 out of 14 sequenced CPT1B 3′ cDNA clones contain part of intron 19 in stead of exon 20. We found that differential polyadenylation is the mechanism behind the existence of these alternative 3′ CPT1B mRNA forms.

Identification ofPorto-1, a new repeated sequence that localises close to the centromere of chromosome2 ofDrosophila melanogaster

We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster.

Characterization of a Translocation-Associated Deletion Defines the Candidate Region for the Gene Responsible for Branchio-Oto-Renal Syndrome

Fluorescencein situhybridization analysis of an 8q translocation breakpoint, dir ins(8)(q24.11;q13.3;q21.13), carried by an individual presenting with Branchio-Oto-Renal (BOR) syndrome, resulted in the identification of an associated deletion. The generation of a YAC contig and the isolation of overlapping recombinant P1 and λ phage clones from the region allowed further characterization of this deletion. Its size was estimated to be between 470 and 650 kb, and it was flanked by the two polymorphic markers D8S1060 and D8S1807. This mapping led us to reevaluate the localization of the gene responsible for BOR syndrome and has now focused the search for the BOR gene to within the limits of this deletion.

The complete nucleotide sequences of the SacBII Kan Domain of the P1 pAD10-SacBII cloning vector and three cosmid cloning vectors: pTCF, svPHEP, and LAWRIST16

The complete nucleotide sequence of the 16,009-bp SacBII Kan domain of the P1 pAD10-SacBII cloning vector and the sequences of three cosmid cloning vectors, pTCF (7941 bp), svPHEP (9201 bp), and LAWRIST16 (5194 bp) have been determined. A modified diatomaceous earth (Prep-A-Gene)-based procedure, which rapidly yields highly supercoiled double-stranded DNA from recombinant P1 and cosmid clones suitable for generating shotgun libraries, also has been developed. The isolated recombinant DNAs were physically sheared to generate 1- to 2-kb fragments that then were blunt-ended and subcloned into double-stranded pUC-based sequencing vectors. The double-stranded sequencing templates were isolated by an alkaline lysis method and subjected to Taq polymerase catalyzed fluorescent end-labeled primer cycle sequencing. After shotgun sequence assembly, contig gaps were closed and ambiguities were resolved via Sequenase catalyzed fluorescent dye-terminator sequencing.

A single P1 clone bearing three genes from human chromosome 11p15.5: HRC1, HRAS1, and RNH

Molecular genetic alterations of chromosome 11p15.5 are a common finding in human cancer. We previously reported the characterization of two cosmids representing a 55-kilobase (kb) region of DNA surrounding the protooncogene HRAS1. A cluster of genes was identified adjacent to this locus, and one of these genes, HRC1, was divergently transcribed 30 kb upstream from HRAS1. A recent report placed the gene for placental ribonuclease inhibitor ( RNH, ribonuclease-angiogenin inhibitor) within 90 kb of HRAS1 by pulsed-field gel electrophoresis (PFGE) mapping. We used recombinant P1 bacteriophage clones for physical mapping to determine the position of RNH relative to the HRAS1 transcription unit and HRC1 on chromosome 11p15.5. PFGE and Southern analysis of genomic DNA suggested the order of the genes ( HRC1-HRAS1-RNH). P1 clones confirmed this assignment, and placed RNH within 30–50 kb of the 3′ end of HRAS1. Furthermore, a single 80-kb P1 clone that bears all three genes was isolated and clarified the Not I restriction map for the HRAS1-RNH interval. Their close physical association was predicted by simple screening of an arrayed P1 library; the clone containing all three genes was selected from multiple positive signals obtained for each HRC1 and RNH because it mapped to the same library-well address.

Purification and Characterization of Recombinant Polymeric Hemoglobin P1 of Glycera dibranchiata

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the α-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).

Transmission of conformational change from the heparin binding site to the reactive center of antithrombin

Heparin greatly increases the rates at which antithrombin inhibits target proteinases. An important part of this rate acceleration is a heparin-induced conformational change in antithrombin. To answer the question of whether or not this change is transmitted to the reactive center, we have prepared a recombinant P1 mutant of antithrombin, R393C, labeled the cysteine with nitrobenzofuran (NBD) fluorophore, and examined the perturbation of NBD fluorescence intensity as a function of bound sulfated oligosaccharide. Two high-affinity heparins, low-affinity heparin, and dextran sulfate were used. We found (i) that binding to antithrombin of all these oligosaccharides resulted in transmission of conformational change to P1 in the reactive center, (ii) that these oligosaccharides all gave enhancements of the rate of inhibition of factor Xa beyond any contribution from surface approximation, and (iii) that the degree of perturbation of P1 correlated with the enhancement of the rate of factor Xa inhibition that was not due to surface approximation.

Identification of a surface-exposed immunodominant epitope on outer membrane protein P1 of Haemophilus influenzae type b

Eight murine monoclonal antibodies (MAbs) directed against outer membrane protein P1 of Haemophilus influenzae type b were generated and characterized. Seven of the eight MAbs reacted with recombinant P1 and purified P1 protein from H. influenzae type b strains MinnA and 1613; MAb P1.8 was specific for the latter strain. A panel of 32 nontypeable and 140 encapsulated Haemophilus strains recovered worldwide representing the major clonal families of serotypes a, b, and d was used to evaluate the distribution among Haemophilus strains of the epitopes identified by the P1-specific MAbs. The epitope reactive with the seven MAbs which recognized P1 from strains MinnA and 1613 was shared by 92% of the encapsulated Haemophilus isolates tested. The epitope is present in the H. influenzae type b strains from clonal families commonly recovered from cases of invasive disease in North America and Europe. A series of nested 5' and 3' deletions of the P1 gene were constructed and analyzed to localize the determinants on P1 recognized by the MAbs. MAbs P1.2, P1.4, P1.5, P1.6, and P1.7 recognized an epitope localized to the carboxy-terminal portion of P1. Murine MAbs P1.1 and P1.3 and two human MAbs, HiH-7 and HiH-10, recognized a complex epitope which was partially localized to the carboxy-terminal portion of the P1 protein. These data indicate that an immunodominant surface-exposed epitope is present on the carboxy-terminal portion of the P1 protein of type b Haemophilus isolates responsible for the majority of invasive disease in North America.