title: Use of the amicyanin signal sequence for efficient periplasmic expression in E. coli of a human antibody light chain variable domain.
abstract: Periplasmic localization of recombinant proteins offers advantages over cytoplasmic protein expression. In this study signal sequence of amicyanin, which is encoded by the mauC gene of Paracoccus denitrificans, was used to express the light chain variable domain of the human κIO8/O18 germline antibody in the periplasm of Escherichiacoli. The expressed protein was purified in good yield (70mg/L of culture) in one step from the periplasmic fraction by affinity chromatography using an engineered hexahistidine tag. Circular dichroism spectroscopy was used to determine if the secondary and tertiary structures of the protein and its thermal stability corresponded to those of the native folded protein. The expressed and purified protein was indeed properly folded and exhibited a reasonable thermal transition temperature of 53°C. These results indicate that the amicyanin signal sequence may be particularly useful for prokaryotic expression of proteins which are prone to mis-folding, aggregation or formation of inclusion bodies, all of which were circumvented in this study.
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32827, United States.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Dow
authors.affiliation: Department of Physics, University of Central Florida, Orlando, FL 32816, United States.
authors.forename: Suren A
authors.initials: SA
authors.lastname: Tatulian
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32827, United States. Electronic address: victor.davidson@ucf.edu.
authors.forename: Victor L
authors.initials: VL
authors.lastname: Davidson
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D010231:Paracoccus denitrificans / Q000235:genetics*; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Amicyanin; Antibody; Protein expression; Protein folding; Signal sequence