title: Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy.
abstract: A dual affinity fusion approach has been used to study the expression and secretion of labile recombinant proteins in Escherichia coli. Here we show that three small eukaryotic proteins (human proinsulin, a thioredoxin homologous domain of rat protein disulfide isomerase, and the extracellular domain of the alpha 1.2-chain of a human T-cell receptor) are stabilized in vivo using a dual affinity fusion strategy, where the gene encoding the desired product is fused between two genes encoding two different affinity domains. Relatively high yields of full-length product were obtained for all three proteins as compared to when fused to a single fusion partner. Despite the use of a signal peptide, significant amounts of the disulfide protein isomerase and T-cell receptor gene products were maintained in the cytoplasm, while the proinsulin fusion was efficiently secreted to the periplasm. Interestingly, the E. coli heat shock proteins DnaK and GroEL were associated with the fusion proteins isolated from the cytoplasm.
authors.affiliation: Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Murby
authors.forename: L
authors.initials: L
authors.lastname: Cedergren
authors.forename: J
authors.initials: J
authors.lastname: Nilsson
authors.forename: P A
authors.initials: PA
authors.lastname: Nygren
authors.forename: B
authors.initials: B
authors.lastname: Hammarberg
authors.forename: B
authors.initials: B
authors.lastname: Nilsson
authors.forename: S O
authors.initials: SO
authors.lastname: Enfors
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases; D051381:Rats; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006360:Heat-Shock Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D004247:DNA; D011384:Proinsulin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases