SRC3 Protein Purification And Structural Analysis

As the core transcription co-activators of the nuclear hormone receptors,three homologous members(SRC1,SRC2 and SRC3)of the p160 family proteins were discovered by scientists in mid of 1990.SRC1(NCOA1)was first cloned in 1995,and soon after SRC2(TIF2,or GRIP1 or NCOA2)and SRC3(also known as P/CIP,RAC3,AIB1,ACTR,TRAM1 and NCOA3)were reported.A large number of literature indicate that members of the SRCs family are not only key transcriptional co-activators for nuclear hormone receptors,but they are also widely involved in transcriptional regulation of many other transcription factors.Furthermore,SRC3 is often amplified and plays a role in cancer.SRC3 and SRC1 were reported to have histone acetyltransferase activity in 1997,but it has since been controversial whether SRC1 and SRC3 are genuine histone acetyltransferases.So far there is a lack of report on SRC3 protein structure,which is likely to provide key information about its enzyme activity.To solve the issue on histone acetyltransferase activity and to provide structural information for developing SRC3 small molecular inhibitors,we wished to determine the structure of SRC3 protein.To this end,we first attempted to use E.coli system for expression and purification of large quantity of different truncated SRC3 proteins.Unfortunately we found that SRC3 proteins were only expressed in low levels in E.coli,regardless the expression vectors or tags used.We then attempted to improve SRC3 expression in E.coli by optimizing SRC3 codon usage.However,codon optimization failed to improve the expression level of SRC3 in E.coli.Considering that poor expression of SRC3 in E.coli could be due to the lack of appropriate post-translation modification(s)required for SRC3 protein stability in E.coli,we switched to insect cell systems for SRC3 expression,but found that the SRC3 proteins expressed in insect cells were present in the form of insoluble inclusion bodies.Finally,we expressed SRC3 proteins in mammalian 293 F cells.Although we successfully expressed and purified SRC3 proteins from 293 F cells,we found that the purified SRC3 proteins were easily precipitated in solution.Since we failed to obtain SRC3 proteins in good quality and quantity in three different expression systems,we surmised that an appropriate interacting partner may be required for SRC3 stability in cells.Therefore,we tried coexpression of SRC3 with CARM1 or p300,the known SRC3 interaction proteins.We found that CARM1 cannot but p300 can promote SRC3 solubility and stability.Altogether,our study explains why so far no structure of SRC3 protein has been reported,and provides useful information for analyzing the crystal structure of SRC3/p300 in the future.