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Improved expression of single-chain antibodies in Ustilago maydis

Paper ID Volume ID Publish Year Pages File Format Full-Text
23012 43409 2014 11 PDF Available
Title
Improved expression of single-chain antibodies in Ustilago maydis
Abstract

•Active scFvs were produced in U. maydis employing unconventional secretion.•A highly active synthetic promoter was established for heterologous gene expression.•A quintuple protease deletion strain exhibits reduced proteolysis.•Optimized strains allow purification of active, full-length protein.

To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.

Keywords
scFv, single chain variable fragment; Gus, β-glucuronidase; FLP, flippase—a site-specific recombinase originating from Saccharomyces cerevisiae; FRT, flippase recombination siteUstilago maydis; Unconventional secretion; Protein expression; Protease; Singl
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Improved expression of single-chain antibodies in Ustilago maydis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 165–175
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us