Abstract
We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691–695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102–107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.
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Abbreviations
- AnTET:
-
anhydrotetracycline
- GFP:
-
green fluorescent protein
- IPTG:
-
isopropyl thiogalactoside
- MBP:
-
maltose-binding protein
- NaCl:
-
sodium chloride
- SUMO:
-
small ubiquitin-like modifiers
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Cabantous, S., Pédelacq, JD., Mark, B. et al. Recent Advances in GFP Folding Reporter and Split-GFP Solubility Reporter Technologies. Application to Improving the Folding and Solubility of Recalcitrant Proteins from Mycobacterium tuberculosis. J Struct Funct Genomics 6, 113–119 (2005). https://doi.org/10.1007/s10969-005-5247-5
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DOI: https://doi.org/10.1007/s10969-005-5247-5