Journal of Structural and Functional Genomics

, Volume 6, Issue 2, pp 113–119

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

  • Stéphanie Cabantous
  • Jean-Denis Pédelacq
  • Brian L. Mark
  • Cleo Naranjo
  • Thomas C. Terwilliger
  • Geoffrey S. Waldo
Article

DOI: 10.1007/s10969-005-5247-5

Cite this article as:
Cabantous, S., Pédelacq, J., Mark, B. et al. J Struct Funct Genomics (2005) 6: 113. doi:10.1007/s10969-005-5247-5

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.

Key words

directed evolutionhigh-throughput screeningprotein foldingsplit proteinsolubility

Abbreviations

AnTET

anhydrotetracycline

GFP

green fluorescent protein

IPTG

isopropyl thiogalactoside

MBP

maltose-binding protein

NaCl

sodium chloride

SUMO

small ubiquitin-like modifiers

Copyright information

© Springer 2005

Authors and Affiliations

  • Stéphanie Cabantous
    • 1
  • Jean-Denis Pédelacq
    • 1
  • Brian L. Mark
    • 1
  • Cleo Naranjo
    • 1
  • Thomas C. Terwilliger
    • 1
  • Geoffrey S. Waldo
    • 1
  1. 1.Bioscience DivisionLos Alamos National LaboratoryLos AlamosUSA