High-level expression, polyclonal antibody preparation and sub-cellular localization analysis of mouse Rhox5 protein

• Published in: Protein expression and purification Vol. 54; no. 2; pp. 247 - 252
• Main Authors: Guo, Fen, Li, Shi-Qian, Chu, Yan-Hui, Huang, Xiao-Feng, Sun, Li-Min, Li, Yue-Qin, Li, Hong-Jian, Zhou, Tian-Hong
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2007
• Subjects: Animals; Base Sequence; Blotting, Western; Cloning, Molecular; Escherichia coli; Escherichia coli - metabolism; Fluorescent Antibody Technique, Indirect; Fusion protein; Homeodomain Proteins - biosynthesis; Homeodomain Proteins - immunology; Mice; Molecular Sequence Data; NIH 3T3 Cells; Polyclonal antibody; Prokaryotic expression; Rabbits; Recombinant Fusion Proteins - biosynthesis; Rhox5; Subcellular Fractions - metabolism; Transcription Factors - biosynthesis; Transcription Factors - immunology; Rhox5; Prokaryotic expression; Fusion protein; Polyclonal antibody
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2007.03.021

Mouse reproductive homeobox on the X chromosome (Rhox) is a novel homeobox gene cluster. Rhox5, also called Pem, belongs to the β subcluster of Rhox. Codon analysis indicated that the cDNA contains 16% of codons rarely used in Escherichia coli. To achieve high-level expression of Rhox5, the coding sequence of Rhox5 was amplified and subcloned into the prokaryotic expression vector pET22b (+) in order to produce 6His-tagged fusion protein in the modified BL21 (DE3) cells, namely Rosetta™2 (DE3) cells. The 6His-tagged Rhox5 was expressed efficiently in Rosetta™2 (DE3), compared with marginal expression in BL21 (DE3). The fusion protein amounted to 16% of the total bacterial proteins after induction with 0.4 mM IPTG for 1.5 h at 37 °C. After purification, Rhox5-6His was used to immunize New Zealand white rabbits following standard protocol. The homemade antiserum could detect both endogenous Rhox5 protein expressed in eukaryotic cells (Cos-7) and exogenous GFP-Rhox5 protein. Furthermore, the antiserum was used to determine the localization of Rhox5 in NIH3T3 cells using an immunofluorescence technique. The results demonstrated that Rhox5 was localized predominantly in the nucleus. Preparation of the anti-Rhox5 polyclonal antibody will facilitate further functional study of Rhox5.