title: Overcoming difficulties on synthesis of cardiac troponin-I.
abstract: Human cardiac troponin-I (cTnI) is one of the most sensitive and specific indicators, used in the diagnosis of myocardial infarction. To produce the protein efficiently, Escherichia coli and Pichia pastoris systems were used. Initial trials for the expression in E. coli were not successful, although different expression vectors with different promoters were tested. This led us to use P. pastoris for the expression. After several trials with two different expression strains of P. pastoris, it was concluded that P. pastoris was also not an optimal expression host for cTnI. Comprehensive analysis of the expression systems indicated that an efficient expression is only possible when the gene is optimized for expression in E. coli. For this purpose, the gene was optimized in-silico, but edited manually afterwards. It was synthesized and cloned into pQE-2 vector. Expression was performed using routine experimental conditions. Thus, cTnI could be efficiently expressed from the optimized gene in E. coli. The expression and purification were practical and may be used for commercial purposes since a total yield of 25µg highly pure protein per milliliter of culture could be obtained. The protein was in its ready-to-use form for many biological applications, including as a standard in diagnostic tests and an antigen for antibody production.
authors.affiliation: a Department of Medical Biology , Kocaeli University Medical School , Umuttepe Kocaeli , Turkey.
authors.forename: S Mert
authors.initials: SM
authors.lastname: Selimoglu
authors.affiliation: b DEKART Proteomics Laboratory, Department of Medical Biology , Kocaeli University Medical School , Umuttepe Kocaeli , Turkey.
authors.forename: Murat
authors.initials: M
authors.lastname: Kasap
authors.affiliation: b DEKART Proteomics Laboratory, Department of Medical Biology , Kocaeli University Medical School , Umuttepe Kocaeli , Turkey.
authors.forename: Gurler
authors.initials: G
authors.lastname: Akpinar
authors.affiliation: c Department of Medical Microbiology , Kocaeli University Medical School , Umuttepe Kocaeli , Turkey.
authors.forename: Aynur
authors.initials: A
authors.lastname: Karadenizli
mesh_terms: D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003062:Codon; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019210:Troponin I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003062:Codon; D018076:DNA, Complementary; D011994:Recombinant Proteins; D019210:Troponin I
keywords: Cardiac troponin-I; Escherichia coli; Pichia pastoris; gene optimization; protein expression; protein purification