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Analysis of bromodomain of BRD-7 gene and its prokaryotic expression
Cong Peng1, Ming Zhou, Qiu-hong Zhang
1Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha 410078, P. R. China.
Background & Objective:
BRD-7 is a novel gene (AF: 152604), containing a bromodomain, was cloned in our lab. Previous studies showed that BRD-7 plays an obviously suppressive role on NPC cell growth. In order to clarify the function mechanism of this gene, we investigate an important motif of BRD-7, the bromodomain.
Methods:
The bromodomain of BRD-7 was analyzed by homology-based amino sequence and secondary structure analysis. In addition, we constructed a prokaryotic expression vector of bromodomain. Western blot analysis was used to confirm the expression of the bromodomain protein in Escherichia coli.
Results:
Homology-based sequence analysis revealed that the bromodomain of BRD-7 possibly contains four alpha helices (Z, A, B and C), and a hydrophobic pocket which is an important structure to recognize acetylated histone peptide. This bromodomain encoding a 12.8 kD protein, was introduced into Escherichia coli using the pGEX-4T-2 expression vector. After isopropyl beta-D-thiogalactopyranoside(IPTG) induction, a new anticipated protein of 38.8 kD appeared on SDS-PAGE and the result was confirmed by Western blot analysis.
Conclusion:
BRD-7 of bromodomain protein is similar to three proteins containing known structural bromodomain motif by bio-informatics analysis, suggesting the bromodomain of BRD-7 should belong to co-activator subgroup and may have similar function that can selectively interact with acetylated histone peptide.