title: Development of CYB5-fusion monitoring system for efficient periplasmic expression of multimeric proteins in Escherichia coli.
abstract: Despite all advances of heterologous expression of recombinant proteins in Escherichia coli, expression of multidomain disulphide-rich proteins faces some problems due to the absence of the possibility to monitor the process in real-time. Here we provide a CYB5-fusion system - cytochrome b5 fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. We validated this system by Fab and scFv antibody fragments expression in order to improve antibody-derived molecules characterization and to simplify their usage. The combination of redox dependent absorbance spectrum of red-colored cytochrome b5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b5 significantly enhances expression level of Fab/scFv antibody fragments in Escherichia coli periplasm.
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevicha 5/2, Minsk, 220141, Belarus.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Dormeshkin
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevicha 5/2, Minsk, 220141, Belarus. Electronic address: agilep@iboch.bas-net.by.
authors.forename: Andrei
authors.initials: A
authors.lastname: Gilep
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevicha 5/2, Minsk, 220141, Belarus.
authors.forename: Gennady
authors.initials: G
authors.lastname: Sergeev
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevicha 5/2, Minsk, 220141, Belarus.
authors.forename: Sergey
authors.initials: S
authors.lastname: Usanov
mesh_terms: D000818:Animals; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006854:Hydrocortisone / Q000037:antagonists & inhibitors / Q000737:chemistry; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D015786:Cytochromes b5; D006854:Hydrocortisone
keywords: Cytochrome b(5); Protein expression monitoring; Protein quantification; Recombinant antibodies