Expression and Application of Recombinant p300 Histone Acetyltransferase Domain

Acetylation of histones is believed to be the key step in transcription initiation.Histone acetyltransferases(HATs) such as p300 catalysed the acetylation of histones and a number of non-histone proteins.HATs appear to be capable of contributing to transcriptional activation,cell cycle progression,gene silencing,DNA repair and other cellular functions.Moreover,acetylation of proteins play a role in nuclear import,protein-protein interactions,protein stability and DNA binding affinity.The histone acetyltransferase p300 acetylates a variety of substrates including transcription factors,signaling regulators and cytoskeletal proteins.Besides,there are also reports concerning the potential of viral proteins to interact with p300 to favor virus replication.Therefore,p300 has been shown to be a versatile transcriptional co-activator in cells.The histone acetyltransferase domain of p300(HAT) is the minimal and central functional domain that bear intrinsic acetyltransferase activity to exert p300-dependent acetylation.In the present study,a recombinant protein corresponding to the HAT coding sequence of p300 was expressed in E.coli BL21(DE3) as a fusion protein with GST(GST-p300 HAT).Histone H3 was incubated with purified p300 HAT domain in the presence of Ac-CoA and detected for acetylation with antibodies against acetylated lysine to assess enzymatic activity.The results suggested that p300HAT domain efficiently acetylated histone H3 in vitro.We further optimize the reaction conditions of in vitro acetylation assay such as reaction buffer,quantity of Ac-CoA and p300 HAT used,and incubation time.In conclusion,we have developed a simple,robust,and non-radioactive in vitro assay for acetylation of histone and non-histone proteins.This assay can be modified easily to acetylate other candidate substrates for investigation of the degree and mechanism of acetylation,as well as the study on the functional advantage of these acetylated proteins.