1. Overexpression of proteins in Escherichia coli (E. coli) can lead to insoluble material (inclusion body) which requires solubilization of the recombinant protein with denaturants and subsequent refolding to the native conformation. This is particularly a problem when proteins are overexpressed at levels suitable for structure analysis by NMR spectroscopy or x-ray crystallography. We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. 2. The Pharmacia BIAcore Biosensor was used to measure the real-time interaction of E. coli ssb protein with poly(deoxythymidylic acid), (dT70). The rationale for an experimental protocol for this type of binding reaction is presented. This approach may have general applicability in the design and analysis of BIAcore binding experiments. 3. The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The in vitro phosphorylation of p51 ETS1 by CAM Kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII) and binding to its specific DNA sequence is not sensitive to calcium signalling.
