A yeast platform for the production of single-chain antibody-green fluorescent protein fusions.
Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 microg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.
Huang
Dagang
D
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Shusta
Eric V
EV
D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region; D007150:Immunohistochemistry / Q000379:methods; D018613:Microscopy, Confocal; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D057127:Single-Chain Antibodies
D007135:Immunoglobulin Variable Region; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C410726:scFV green fluorescent protein, recombinant; D049452:Green Fluorescent Proteins