title: Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics.
abstract: The proteomics technique, which consists of two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), gel image analysis, and multivariate statistics, was applied to the phase analysis of a fed-batch culture for the production of a single-chain variable fragment (scFv) of an anti-C-reactive protein (CRP) antibody by Pichia pastoris. The time courses of the fed-batch culture were separated into three distinct phases: the growth phase of the batch process, the growth phase of the fed-batch process, and the production phase of the fed-batch process. Multivariate statistical analysis using 2-DE gel image analysis data clearly showed the change in the culture phase and provided information concerning the protein expression, which suggested a metabolic change related to cell growth and production during the fed-batch culture. Furthermore, specific proteins, such as alcohol oxidase, which is strongly related to scFv expression, and proteinase A, which could biodegrade scFv in the latter phases of production, were identified via the PMF method. The proteomics technique provided valuable information about the effect of the methanol concentration on scFv production.
authors: Department of Chemistry and Materials, Kyoto Institute of Technology, Goshokaidocho, Sakyoku, Kyoto 606-8585, Japan; Department of Bio-molecular Engineering, Kyoto Institute of Technology, Goshokaidocho, Sakyoku, Kyoto 606-8585, Japan.
authors: Yuya
authors: Y
authors: Fujiki
authors: Department of Chemistry and Materials, Kyoto Institute of Technology, Goshokaidocho, Sakyoku, Kyoto 606-8585, Japan; Department of Bio-molecular Engineering, Kyoto Institute of Technology, Goshokaidocho, Sakyoku, Kyoto 606-8585, Japan.
authors: Yoichi
authors: Y
authors: Kumada
authors: Department of Chemistry and Materials, Kyoto Institute of Technology, Goshokaidocho, Sakyoku, Kyoto 606-8585, Japan; Department of Bio-molecular Engineering, Kyoto Institute of Technology, Goshokaidocho, Sakyoku, Kyoto 606-8585, Japan. Electronic address: kishimoto@kit.jp.
authors: Michimasa
authors: M
authors: Kishimoto
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D002097:C-Reactive Protein / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D000432:Methanol / Q000494:pharmacology; D015999:Multivariate Analysis; D010449:Peptide Mapping; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D002097:C-Reactive Protein; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II; D000432:Methanol
keywords: Cancerd2; Peptide mass fingerprinting; Phase analysis; Pichia pastoris; Proteomics; Single-chain variable fragment; Two-dimensional gel electrophoresis