Definition of the domain boundaries is critical to the expression of the nucleotide-binding domains of P-glycoprotein.
Heterologous expression of domains of eukaryotic proteins is frequently associated with formation of inclusion bodies, consisting of aggregated mis-folded protein. This phenomenon has proved a significant barrier to the characterization of domains of eukaryotic ATP binding cassette (ABC) transporters. We hypothesized that the solubility of heterologously expressed nucleotide binding domains (NBDs) of ABC transporters is dependent on the definition of the domain boundaries. In this paper we have defined a core NBD, and tested the effect of extensions to and deletions of this core domain on protein expression. Of 10 NBDs constructed, only one was expressed as a soluble protein in Escherichia coli, with expression of the remaining NBDs being associated with inclusion body formation. The soluble NBD protein we have obtained corresponds to residues 386-632 of P-glycoprotein and represents an optimally defined domain. The NBD has been isolated and purified to 95% homogeneity by a two-step purification protocol, involving affinity chromatography and gel filtration. Although showing no detectable ATP hydrolysis, the protein retains specific ATP binding and has a secondary structure compatible with X-ray crystallographic data on bacterial NBDs. We have interpreted our results in terms of homology models, which suggest that the N-terminal NBD of P-glycoprotein can be produced as a stable, correctly folded, isolate domain with judicious design of the expression construct.
Kerr
Ian D
ID
School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK. ian.kerr@nottingham.ac.uk
Berridge
Georgina
G
Linton
Kenneth J
KJ
Higgins
Christopher F
CF
Callaghan
Richard
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009711:Nucleotides; D000255:Adenosine Triphosphate