期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 416, 期 1, 页码 94-107出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2011.12.021
关键词
antibody engineering; bacterial surface display; directed evolution; protein expression and folding; twin-arginine translocation protein export pathway
资金
- National Science Foundation [CBET-0449080]
- New York State Office of Science, Technology and Academic Research Distinguished Faculty
- National Institutes of Health [R41GM090585]
- National Cancer Institute [F32CA150622]
A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process. (C) 2011 Elsevier Ltd. All rights reserved.
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