Molecular characterization of proteolytic processing of the Gag proteins of human spumaretrovirus.
Molecular characterization of proteolytic processing of the human spumaretrovirus (HSRV) Gag proteins and the precise determination of cleavage sites was performed. For in vitro processing of recombinant HSRV Gag proteins, a recombinant enzymatically active HSRV protease was employed. Recombinant Gag proteins and protease were cloned and expressed as hexa-histidine-tagged proteins in pET-32b and pET-22b vectors, respectively, in the E. coli BL21 expression strain. The recombinant proteins were purified by affinity chromatography on an immobilized metal ion matrix. To determine the precise processing sites, recombinant Gag proteins or synthetic peptides derived from Gag sequences were cleaved in vitro by the recombinant protease. Proteolytic processing reactions were carried out under optimal reaction conditions of HSRV protease in sodium phosphate buffer, pH 6.0, supplied with 2 M NaCl at 37 degrees C. The cleavage sites were determined by amino-terminal amino acid sequencing as well as by matrix-assisted laser desorption/ionization mass spectrometry analysis of the reaction products. Fluorescence spectrophotometry was used to determine cleavage kinetics of peptides mimicking different cleavage sites within the HSRV Gag proteins.
Pfrepper
Klaus-Ingmar
KI
Department of Research and Development, Mikrogen GmbH, Martinsried, Germany.
Flügel
Rolf M
RM
D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D015531:Radioimmunoprecipitation Assay / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D016092:Spumavirus / Q000378:metabolism*
D015683:Gene Products, gag; D011994:Recombinant Proteins; D010447:Peptide Hydrolases