title: Process optimization for increased yield of surface-expressed protein in Escherichia coli.
abstract: The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.
authors: Division of Industrial Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), SE 106 91, Stockholm, Sweden, johanjar@kth.se.
authors: Johan
authors: J
authors: Jarmander
authors: Lars
authors: L
authors: Janoschek
authors: Susanna
authors: S
authors: Lundh
authors: Gen
authors: G
authors: Larsson
authors: Martin
authors: M
authors: Gustavsson
mesh_terms: D048168:Escherichia coli K12 / Q000235:genetics / Q000254:growth & development*; D036781:Fimbriae Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D019779:Salmonella enterica / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D036781:Fimbriae Proteins