Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: Comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein

Changsen Wang, Ariel F. Castro, Denise M. Wilkes, Guillermo A. Altenberg

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multi-spanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.

Original languageEnglish (US)
Pages (from-to)77-81
Number of pages5
JournalBiochemical Journal
Volume338
Issue number1
DOIs
StatePublished - Feb 15 1999

Keywords

  • Cancer
  • Cystic fibrosis
  • Fusion proteins
  • Multidrug resistance
  • P-glycoprotein

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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