Chemical synthesis of peptides and proteins.
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Peptides↗
Biomedical subjects
S B Kent
Publications and source records attributed to S B Kent.
Isolation and characterization of a corticotropin-releasing hormone-like peptide from human placenta.
Immunoreactive CRH was detected in extracts of human term placentae [5.2 +/- 0.8 (+/- SE) pmol/g wet wt; n = 9]. Molecular sieve chromatography revealed three size classes of immunoreactive CRH. The major species eluted with the Kav of synthetic rat CRH; the minor species had apparent mol wt (MW) of 18,000 and 8,000. A placental CRH-(1-41)-sized peptide was isolated by fractional acetone precipitation, molecular sieve chromatography, and sequential reverse phase high performance liquid chromatography steps. This peptide had the same chromatographic behavior as did rat CRH in all high performance liquid chromatographic isolation steps as well as the same UV absorbance to immunoreactive CRH ratio after the final purification step. Purified placental CRH stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with synthetic rat CRH. Partial sequencing indicated that 32 amino acids of this peptide are identical to those of rat and human CRH (sequence deduced from genomic sequence), and comparative peptide mapping with rat CRH provided further evidence that the placental CRH-like peptide is very homologous if not identical to CRH. The high mol wt placental CRH fractions also were partially purified by acetone precipitation, immune affinity chromatography, and gel filtration. Neither of these materials [big form (MW, 18,000) or intermediate form (MWr, 8,000)] stimulated ACTH release from rat pituitary tissue in vitro. Limited trypsin digestion of the highest MW CRH, followed by gel filtration analysis, resulted in conversion to the smaller [8,000 MW-sized and CRH-(1-41)-sized] forms. The detection of a CRH-like peptide in placenta together with our previous demonstration of plasma immunoreactive CRH in pregnant women suggest that the placenta synthesizes and secretes CRH into the maternal circulation.
Amino Acid Sequence↗
Antipeptide antibodies of predetermined specificity recognize and neutralize the bioactivity of the pan-specific hemopoietin interleukin 3.
Ten peptides that corresponded to portions of the T cell lymphokine pan-specific hemopoietin interleukin 3 (IL 3) were synthesized, coupled to keyhole limpet hemocyanin, and used to raise antipeptide antibodies in rabbits. These antisera reacted to varying degrees with native biologically active IL 3. Antibodies directed against peptides corresponding to residues 1-29 at the NH2 terminus, 123-140 at the COOH terminus, and to residues 64-82 and 91-112 were affinity-purified on peptide columns. Immunoabsorbent columns produced from affinity-purified antibodies to the 1-29, 91-112, and 123-140 although not the 64-82 peptide were effective in depleting biologically active IL 3 from conditioned medium. However, the antibodies specific for peptides 91-112 and 123-140 had only a low affinity for native IL 3 and it was only in the case of the anti-1-29 antibodies that a significant amount of IL 3 remained bound after extensive washing and could be recovered from the column by acid elution. The affinity-purified antibodies directed to peptides 1-29, 91-112, and 123-140 significantly inhibited the biological activity of IL 3, although with different dose-response characteristics. Anti-1-29 antibodies inhibited bioactivity over a wide range of concentrations (down to 20 ng/ml) although the inhibition was never complete. In contrast, the anti-91-112 antibodies, although effective only at high concentrations, produced complete inhibition of biological activity. These experiments demonstrated that antibodies to defined peptides can be used to generate antibodies to native IL 3 and should form useful tools in analyzing the structure and function of the native molecules.
Animals↗
Antipeptide antibodies define the NH2-terminal structure of the pan-specific hemopoietin interleukin 3.
Antipeptide antibodies were raised in rabbits against a synthetic peptide including the six NH2-terminal amino acids of the pan-specific hemopoietin interleukin 3 (IL 3). Affinity-purified antibody preparations specific for epitopes determined by residues 1 to 6 were immobilized and used as affinity columns. Up to 98% of IL 3 bioactivity in T cell-conditioned medium was depleted by these columns, as was 71 and 74%, respectively, of IL 3 aberrantly produced by the myeloid leukemias WEHI-274.14 and WEHI-3B. IL 3 produced in vivo in WEHI-3B tumor-bearing mice also bound to the anti-1-6 antibody column, up to 70% of the bioactivity being depleted from ascites fluid and up to 84% from the serum. These results suggest that all IL 3 secreted by T cells and the majority of the IL 3 molecules secreted by myeloid leukemias express epitopes determined by residues 1-6 and cannot have the NH2-terminal amino acid sequence initially reported for IL 3. These six NH2-terminal amino acids share similarities with the NH2-terminal amino acids of several other lymphokines, suggesting an important function for this hexapeptide.
Amino Acid Sequence↗
Antibodies to synthetic peptides from the pre-S1 and pre-S2 regions of one subtype of the hepatitis B virus (HBV) envelope protein recognize all HBV subtypes.
Immunodominant B and T cell epitopes have been demonstrated recently on the preS1 and PreS2 regions of the hepatitis B virus (HBV) envelope protein. Synthetic peptide analogs corresponding to the preS2 region elicit virus-neutralizing antibodies and protect chimpanzees against HBV infection. Antibodies raised by immunization with peptides derived from the preS1 sequence block the site involved in HBV attachment to cell receptors, and are expected to be virus-neutralizing. Results presented here show that antisera raised against synthetic peptide analogs carrying the immunodominant epitope of the preS1 and preS2 sequence, respectively, and corresponding to two HBV subtypes, adw2 and ayw, each recognized preS1 and preS2 specific epitopes on all serological subtypes of the HBV envelope protein. Thus, the sequence variability within the preS1 and preS2 regions does not represent an impediment to the development of synthetic peptide or genetically engineered hepatitis B preS immunogens for worldwide immunization.
Amino Acid Sequence↗
Immunological cross-reactivity between preS2 sequences of the hepatitis B virus envelope proteins corresponding to serological subtypes adw2 and ayw.
An immune response to epitopes localized on the preS region of the hepatitis B virus (HBV) envelope (env) is elicited during recovery from HBV infection and appears to play a role in virus clearance. Anti-preS antibodies (Ab) are expected to be protective against HBV infection as indicated by the virus-neutralizing capacity of Ab to a preS2-specific synthetic peptide preS(120-145). However, there is considerable amino acid variability between preS regions corresponding to distinct serological subtypes of HBV, raising the question whether the preS sequences are sufficiently related immunologically to have the potential of inducing cross-protective immunity. To answer this question concerning the preS2 region, antisera to synthetic peptides preS(120-153) and preS(128-153) corresponding to subtypes adw2 and ayw, as well as to the native env [ayw] protein were raised. Using the resulting polyclonal Ab, an immunological relatedness between preS2 sequences of subtypes adw2 and ayw was demonstrated. On the other hand, Ab selected by affinity chromatography or by cloning hybridoma cells may recognize with strong preference subtype-specific determinants within the preS2 region when the compared antigens are in solution rather than on the solid phase. These findings have implication for the design of: (1) preS2-specific immunogens and (2) immunoassays for quantitation of preS2 sequences in HBV env proteins.
Amino Acid Sequence↗
Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.
We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from one- and two-dimensional polyacrylamide gels.
Amino Acid Sequence↗
E-domain peptide of rat proinsulin-like growth factor-II: validation of a radioimmunoassay and measurement in culture medium and rat serum.
We recently discovered a peptide derived from the carboxyl-terminal portion of the E-domain of rat proinsulin-like growth factor II (pro-IGF-II) in medium conditioned by BRL-3A rat liver cells. This peptide begins at residue 117 in the pro-IGF-II sequence. To measure physiological concentrations of this peptide in serum, we established an RIA for a synthetic peptide [rat pro-IGF-II-(117-156); E-domain peptide] corresponding to the carboxyl-terminal 40-amino acids of rat pro-IGF-II. The 41-residue peptide [Tyr116]pro-IGF-II-(117-156) was also synthesized and iodinated for use as tracer. Using polyclonal antibodies, we established a standard curve that measured as little as 25 pg/tube. Tracer was not displaced by insulin, human (h) IGF-I, hIGF-II, pro-hIGF-I-(71-105), rat GH, mouse EGF, ACTH, bovine PTH, ovine FSH, TRH, or LHRH under our assay conditions. However, a synthetic analog of the E-domain peptide [Phe117]pro-IGF-II-(118-156) showed displacement similar to that of the synthetic E-domain peptide. Serial dilutions of either culture medium or rat serum exhibited displacement parallel to the standard curve. Measurement of E-domain peptide in serum-free medium conditioned by BRL-3A rat liver cells showed a time-related increase in E-peptide concentration over a 72-h period. Analysis of E-peptide immunoreactivity from conditioned medium after gel filtration chromatography in 1 M acetic acid revealed a single peak which had a mol wt (determined by Western blot) identical to that of the synthetic E-peptide standard. The concentration of immunoreactive E-domain peptide levels in serum of 5-day-old rat pups was 30-40 times higher than concentrations in the serum of adult rats. Gel filtration chromatography of adult rat serum in 1 M acetic acid revealed a single major peak of immunoreactivity eluting at a position similar to the elution position of the E-domain peptide from BRL-3A rat liver cell-conditioned medium. The RIA described here should prove useful for measurement of the somatic output of E-domain peptide under different physiological conditions.
Animals↗
Production of antibodies recognizing a hepatitis B virus (HBV) surface antigen by administration of murabutide associated to a synthetic pre-S HBV peptide conjugated to a toxoid carrier.
The influence of Murabutide, a muramyl peptide analog, on the response of mice to two synthetic hepatitis B virus antigens was compared with the activity of Al (OH)3 and of FCA. The synthetic peptides represented fragments either of the major component of the hepatitis B surface antigen (HBsAg) or of the pre-S region. They were used either conjugated to toxoid carrier or as a totally synthetic preparation, i.e. copolymerized with a streptococcal peptide. Our results demonstrated that treatment with Murabutide increased the levels of antibodies, modulated their specificity and allowed a better recognition of the natural antigens.
Acetylmuramyl-Alanyl-Isoglutamine↗
Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus.
Hepatitis B virus (HBV) has not yet been propagated in vitro, and knowledge concerning its reaction with receptors on target cells remained scant. We have located within the HBV envelope proteins a sequence mediating the attachment of HBV to human hepatoma HepG2 cells. A synthetic peptide analog (PLGFFPDHQLDPAFGANSNNPDWDFNP) is recognized by both cell receptors and anti-HBV antibodies and elicits antibodies reacting with native HBV. The synthetic peptide is a promising immunogen expected to elicit protective antibodies based on the concept of the attachment blockade pathway of virus neutralization. The approach described here, based on anti-peptide antisera and the binding of peptide analogs to cell receptors is generally applicable for the delineation of cell receptor binding sites on viruses with known gene sequences.
Amino Acid Sequence↗
Immune response to the pre-S(1) region of the hepatitis B surface antigen (HBsAg): a pre-S(1)-specific T cell response can bypass nonresponsiveness to the pre-S(2) and S regions of HBsAg.
Hepatitis B surface antigen (HBsAg) particles are composed of a major polypeptide, p25, and additional polypeptides of higher m.w., namely p33 and p39, are variably present. All three polypeptides share the 226 amino acid residues of the S region: p33 consists of the p25 sequence plus an NH2-terminal 55 residues (pre-S(2], and p39 consists of the p33 sequence plus an NH2-terminal 108-119 residues (pre-S(1). In previous studies we demonstrated the influence of two Ir genes on the humoral and cellular immune responses to the S region and identified nonresponder phenotypes (H-2f,s). Subsequent studies showed that the immune response to the pre-S(2) region was regulated by H-2-linked genes independently of the S region response, such that immunization of S region nonresponder, pre-(S2) region responder mice (H-2s) with HBsAg/p33 circumvented nonresponse to the S region. In the present study, we have extended this analysis to the pre-S(1) region of HBsAg, with the following results: 1) and pre-S(1) region is immunogenic at the T and B cell levels; 2) anti-pre-S(1) specific antibody production is regulated by H-2-linked genes and can be independent of anti-S and anti-pre-S(2) antibody production; 3) immunization of H-2f strains with HBsAg/p39 particles containing the pre-S(1) region can bypass nonresponsiveness to the S and pre-S(2) regions in terms of antibody production; 4) two synthetic peptides, p32-53 and p94-117, define murine and human antibody binding sites on the pre-S(1) region, and p1-21 and p12-32 define additional human antibody binding sites; 5) pre-S(1)-specific T cells can be elicited in S and pre-S(2) region nonresponder mice (H-2f) and provide functional T cell help for S-pre-S(2)-, and pre-S(1)-specific antibody production; and 6) a T cell recognition site in the pre-S(1) region, p12-32 was identified. These results are relevant to HBV vaccine development, and possibly to viral clearance mechanisms, since the higher m.w. polypeptides are preferentially expressed on intact virions.
Acute Disease↗
Microscale structure analysis of a high-molecular-weight, hydrophobic membrane glycoprotein fraction with platelet-derived growth factor-dependent kinase activity.
General methods for the study of the primary structure of picomole quantities of large, hydrophobic membrane glycoproteins with blocked amino-termini have been developed. Three techniques designed to be used in concert with each other are described: first, modified protein preparation and fragmentation techniques; secondly, a simple but very selective two-dimensional reversed-phase high-performance liquid chromatography system for the resolution of complex mixtures of small to medium-sized tryptic peptides on Vydac C4, C18 and diphenyl columns and thirdly, a two-dimensional separation method for large, denaturated (CNBr) polypeptide fragments by size-exclusion high-performance liquid chromatography, combined with either reversed-phase high-performance liquid chromatography (C4) or sodium dodecyl sulphate polyacrylamide gel electrophoresis in conjunction with electroblotting and autoradiography. These methods were applied to studies of the platelet-derived growth factor receptor. Starting with 500 pmoles of purified protein, a total of 232 amino acids were sequenced.
Alkylation↗
Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis.
We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein-containing bands or spots are cut out and inserted directly into a gas-phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150-pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels.
Amino Acid Sequence↗
Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3.
Interleukin-3 (IL-3), a protein of 140 amino acids, was chemically synthesized by means of an automated peptide synthesizer and was shown to have the biological activities attributed to native IL-3. Assays of synthetic analogues established that an amino terminal fragment has detectable IL-3 activity, but that the stable tertiary structure of the complete molecule was required for full activity. The results demonstrate that automated peptide synthesis can be applied to the study of the structure and function of proteins.
Amino Acid Sequence↗
Characterization of monoclonal antibodies specific for the pre-S2 region of the hepatitis B virus envelope protein.
Monoclonal antibodies (McAb) specific for the pre-S region of the hepatitis B virus (HBV) envelope protein were prepared using HBV particles of hepatitis B surface antigens (HBsAg) as immunogens. The antibodies reacted in Western blot analyses and in ELISA with pre-S2 sequences of the HBV envelope protein. Pepsin or protease V8 treatment of the antigen abolished reactivity. The fine specificity of one of the McAb (F376) was established by immunoassays using synthetic peptides and a pre-S2-beta-galactosidase fusion protein expressed in E. coli. The shortest peptide recognized by F376 is demarcated by residues pre-S(132) at the N-terminal and pre-S(140)-pre-S(145) at the C-terminal. The corresponding amino acid sequence (for HBV subtype adw2) is: QDPRVRGLY(LPAGG). Additional amino acid residues at the N-terminal, and possibly at the C-terminal ends contribute to the binding of McAb, probably due to conformational influences. The McAb was applied to immunoassays of pre-S2 sequences in purified HBsAg and in human sera containing HBsAg.
Antibodies, Monoclonal↗
Antibodies to a synthetic peptide from the preS 120-145 region of the hepatitis B virus envelope are virus neutralizing.
Studies with synthetic peptides have provided evidence for the presence of preS coded sequences in the envelope (env) proteins of hepatitis B virus (HBV) and indicated that these sequences are involved in the specific attachment of HBV to liver cells. Scanning of the preS sequence for immunodominant continuous epitopes identifies the sequence within residues preS (120-145) as the most immunogenic in eliciting antibodies recognizing HBV and as the most efficiently binding antibodies from sera of rabbits and humans immunized with HBV env proteins. To assess the potential of preS (120-145) as a synthetic vaccine against hepatitis B, in vitro neutralization of the virus by rabbit antiserum to the peptide was assayed in chimpanzees. The animals, subsequently proven to be susceptible to HBV infection, did not develop hepatitis B as judged by negative serological tests for HBV-associated antigens and antibodies and by normal serum alanine aminotransferase levels and normal liver biopsies. These results establish the role of preS domains in the process of virus neutralization and the potential of synthetic preS analogues for hepatitis B vaccination.
Animals↗
Scrapie and cellular prion proteins share polypeptide epitopes.
Purified preparations of scrapie prions contain one major protein, PrP 27-30, and aggregates of rod-shaped structures. On the basis of the NH2-terminal amino acid sequence of PrP 27-30, a synthetic peptide (PrP-P1) was constructed. Monospecific rabbit antisera to PrP-P1 were found by immunoblotting to react with PrP 27-30 and its precursor (PrP 33-35Sc), as well as with a related protease-sensitive cellular homologue (PrP 33-35C). An enzyme-linked immunosorbent assay showed that rabbit antiserum to PrP 27-30 was more reactive with PrP 27-30 than with PrP-P1; conversely, antiserum to PrP-P1 was more reactive with the peptide than with the prion proteins. In addition, antibodies to PrP-P1 decorate purified prion rods and stain amyloid plaques in scrapie-infected hamster brain. The peptide epitopes shared by PrP 27-30, PrP 33-35Sc, and PrP 33-35C clearly establish a relationship among these three proteins.
Amyloid↗
Detection of antiviral antibodies with predetermined specificity using synthetic peptide--beta-lactamase conjugates: application to antibodies specific for the preS region of the hepatitis B virus envelope proteins.
Amino acid sequences coded for by the preS region of the hepatitis B virus (HBV) envelope gene are present both in HBV and in subviral hepatitis B surface antigen (HBsAg) particles. Consequently, anti-preS-specific antibodies are elicited during the course of HBV infection. Such antibodies are virus-neutralizing. Therefore, it is important to determine whether or not vaccination with HBsAg also induces an anti-preS-specific immune response. We describe here an enzyme-linked immunosorbent assay applicable for the screening of sera from vaccinated individuals for anti-preS antibodies. IgG from serum specimens was adsorbed to staphylococcal Protein A on a superparamagnetic support and subsequently mixed with a synthetic peptide analogue [preS(120-145)] covalently linked to beta-lactamase. The presence of anti-preS in serum specimens resulted in binding of the conjugated beta-lactamase to the magnetic support. The adsorbed enzyme was quantified colorimetrically.
Amino Acid Sequence↗