Meetei A R, Rao M R
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Protein Expr Purif. 1996 Dec;8(4):409-15. doi: 10.1006/prep.1996.0118.
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.
大鼠精子细胞蛋白TP2是一种碱性核蛋白，每个分子结合两个锌原子。我们在此报告通过RT-PCR方法克隆编码大鼠TP2的互补DNA。克隆的TP2 cDNA的核苷酸序列在几个位置上与文献中已报道的序列不同。我们已将大鼠TP2 cDNA克隆到表达载体pTrc 99A中。用1 mM IPTG诱导后，TP2有低水平表达，且可以以可溶形式回收。重组TP2从可溶形式中纯化出来。使用镍琼脂糖和肝素琼脂糖色谱法从大肠杆菌的可溶提取物中纯化重组TP2，并用抗大鼠TP2抗体进行免疫印迹和放射性65Zn印迹显示其与天然大鼠TP2相同。
