A scFv antibody mutant isolated in a genetic screen for improved export via the twin arginine transporter pathway exhibits faster folding.
Proteins destined for export across the cytoplasmic membrane via the post-translational Sec-dependent route have to be maintained in a largely unfolded state within the cytoplasm. In sharp contrast, only proteins that have folded into a native-like state within the cytoplasm are competent for export via the twin arginine translocation (Tat) pathway. Proteins that contain disulfide bonds, such as scFv antibody fragments, can be translocated via Tat only when expressed in Escherichia coli trxB gor mutant strains having an oxidizing cytoplasm. However, export is poor with the majority of the protein accumulating in the cytoplasm and only a fraction exported to the periplasmic space. Using a high throughput fluorescence screen, we isolated a mutant of the anti-digoxin 26-10 scFv from a large library of random mutants that is exported with a higher yield into the periplasm. In vitro refolding experiments revealed that the mutant scFv exhibits a 250% increase in the rate constant of the critical second phase of folding. This result suggests that Tat export competence is related to the protein folding rate and could be exploited for the isolation of faster folding protein mutants.
Ribnicky
Brian
B
Department of Chemical Engineering, University of Texas, Austin, TX 78712, USA.
Van Blarcom
Thomas
T
Georgiou
George
G
D001120:Arginine / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D013997:Time Factors
D004220:Disulfides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D026901:Membrane Transport Proteins; D001120:Arginine; D010100:Oxygen