Isolation and Properties of Recombinant Human High Mobility Group I/Y Chromosomal Protein

ABSTRACT

Recombinant human high mobility group chromosomal protein I/Y has been expressed in E. coli BL21(DE3) strain using the expression system of Studier et al. (1990). Cells were transformed with bacterial plasmid pET15bHMG-I/Y containing cDNA of the human HMG I/Y protein inserted in-frame with a sequence coding six cnosecutive histidines at the N-terminus of the protein (Thanos and Maniatis, 1992). The recombinant protein has been isolated from the total protein extract by selective binding of the 6xHis-tag on Ni²⁺-NTA resin (Qiagen) using metalo belate affinty chromatography. The protein was tested by SDS polyacrylamide gel electrophoresis and immunochemical detection with anti rat brain HMG I antibodies (IgG fraction). The immunoperoxidase test was performed on Western blots with 4-chloro-l-naphtol/H₂O₂ as a substrate and on dot blots with luminol as an enhanced hemiluminiscence emitting substrate (ECL—Amersham Life Sci.). By its electrophoretic and immunochemical reactions the obtained recombinant human HMG I/Y protein was identical with a natural HCIO₄ extracted rat brain HMG I chromosomal protein.