Efficient expression of the Paramecium calmodulin gene in Escherichia coli after four TAA-to-CAA changes through a series of polymerase chain reactions.
We have expressed the Paramecium calmodulin gene in Escherichia coli by changing the four TAA codons in this gene to CAAs. This was carried out by three polymerase chain reactions (PCRs) and then cloning the product into the expression vector pKK223-3 immediately downstream of its trp-lac hybrid promoter. JM109 strain of E. coli, transformed with the recombinant plasmid harboring the altered Paramecium calmodulin gene, produces a protein judged to be calmodulin. It is recognized by a monoclonal antibody to Paramecium calmodulin; it migrates with the native protein at nearly the same rate in electrophoreses; and it shows a Ca(2+)-dependent shift in electrophoretic pattern. The production of calmodulin is about 170 times as efficient with E. coli as with Paramecium in terms of unit volume of packed cells, and is about 400 times as efficient in unit volume of liquid culture. This method appears useful in site-directed mutageneses and in the heterologous productions of other ciliate proteins. A critique of this method is provided. A calmodulin half-molecule, a by-product of this project, is described.
Department of Genetics, University of Wisconsin, Madison 53706.
J A
JA
Kink
M E
ME
Maley
K Y
KY
Ling
J A
JA
Kanabrocki
C
C
Kung
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003062:Codon; D016054:DNA, Protozoan; D003854:Deoxyribonucleotides; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010247:Paramecium / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification; D013728:Terminator Regions, Genetic / Q000235:genetics*
D002147:Calmodulin; D003062:Codon; D016054:DNA, Protozoan; D003854:Deoxyribonucleotides; D015800:Protozoan Proteins