Cloning of cDNA encoding rat spermatidal protein TP2 and expression in Escherichia coli.
Rat spermatidal protein TP2 is a basic nuclear protein containing two atoms of zinc bound per molecule. We report here cloning of complementary DNA encoding rat TP2 by the RT-PCR method. The nucleotide sequence of cloned TP2 cDNA differs at a few positions from the sequence already reported in the literature. We have cloned rat TP2 cDNA into the expression vector pTrc 99A. Upon induction with 1 mM IPTG, there was a low level of expression of TP2 which could be recovered in the soluble form. Recombinant TP2 was purified from the soluble form. Recombinant TP2 was purified from the soluble extract of E. coli using nickel-agarose and heparin-agarose chromatography and was shown to be identical to native rat TP2 as revealed by immunoblotting with anti-rat TP2 antibodies and radioactive 65Zn-blotting.
Meetei
A R
AR
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Rao
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D015032:Zinc
D002868:Chromosomal Proteins, Non-Histone; D018076:DNA, Complementary; C053886:spermatid transition proteins; D015032:Zinc