Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli.
Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein.
Milner
R E
RE
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Busaan
J
J
Michalak
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D016189:Dystrophin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics; D013205:Staphylococcal Protein A / Q000235:genetics
D001426:Bacterial Proteins; D016189:Dystrophin; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D005982:Glutathione Transferase; C063842:dnaK protein, E coli