[Preparation of the myristoylated and nonmyristoylated form of recombinant recoverin in E. coli cells and comparison of their functional activity].
A recombinant plasmid was constructed for expressing a gene for bovine recoverin under the control of the lac promoter. Coexpression of the recoverin and N-myristoyl transferase genes was performed to prepare recombinant myristoylated recoverin. The obtained systems provide high levels of biosynthesis of the recombinant recoverins in the E. coli cells. Using a reconstructed system, containing urea-washed rod outer segment membranes, purified rhodopsin kinase (RK), and a recoverin, it was shown that the three recoverin forms (natural, recombinant nonmyristoylated, and recombinant myristoylated ones) perform the calcium-dependent regulation of the activity of RK with half a maximum effect at a free calcium concentration of 2 microM. Interestingly, the N-terminal myristoylation of recoverin increased substantially its functional activity.
Zargarov
A A
AA
Senin
I I
II
Alekseev
A M
AM
Shul'ga-Morskoĭ
S V
SV
Filippov
P P
PP
Lipkin
V M
VM
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005136:Eye Proteins / Q000302:isolation & purification; D051606:G-Protein-Coupled Receptor Kinase 1; D051597:Hippocalcin; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D051603:Recoverin
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D051603:Recoverin; D051597:Hippocalcin; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D002118:Calcium