Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase.
A gene coding for the Nereis sarcoplasmic calcium-binding protein (NSCP) was synthesized and expressed in Escherichia coli. The sequence of the gene was derived from the protein sequence by reverse translation. It possesses a number of unique, regularly spaced, restriction endonuclease cleavage sites to facilitate future site-directed mutagenesis. For the cloning strategy the gene sequence was divided into four parts. Three parts were cloned by ligation of hybridized oligomers and one part by inverse PCR. The protein was expressed as a fusion protein with the bacterial chloramphenicol acetyl-transferase (CAT), which could be easily purified by affinity chromatography. At the junction of the CAT and NSCP moieties a recognition site for the proteolytic enzyme factor Xa was built in. However, the distance between the moieties appeared to be crucial to warrant cleavage. A kinetic analysis showed that NSCP prepared from the sandworm and the one expressed by E. coli behaved in the same way. This system provides a basis for site-specific mutagenesis studies, in order to elucidate the molecular mechanism of cation binding and concomitant conformational changes.
Dekeyzer
N
N
Laboratory of Chemical and Biological Dynamics, University of Leuven, Belgium.
Engelborghs
Y
Y
Volckaert
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005796:Genes; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011077:Polychaeta / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D002135:Calcium-Binding Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase