Spectral enhancement of recombinant proteins with tryptophan analogs: The soluble domain of human tissue factor

Abstract

Publisher Summary Tryptophan (Trp) analogs in proteins have great potential for fluorescence studies, provided they have a unique absorbance compared to Trp, allowing proteins incorporating these analogs to be selectively excited in the presence of other Trp-containing proteins. This chapter discusses attempts to replace the four Trps in the truncated, soluble domain of recombinant human tissue factor (sTF) with 5-hydroxytryptophan (5-OHTrp) and 7-azatryptophan (7-ATrp). Among possible Trp analogs, 7-ATrp is an excellent environmental probe, as it can exhibit a large increase in quantum yield, when buried in the protein interior and has approximately 50 nm red-shifted emission spectra compared to Trp, while the analog 5-OHTrp has the advantage of a large shift in the absorbance spectrum, allowing for its selective excitation in the presence of Trp. The chapter describes that cells exposed to 5-OHTrp grow poorly, and express less protein than cells induced in the presence of Trp. To date most of the proteins expressed with 5-OHTrp or 7-ATrp have been prokaryotic, and the prokaryotic DNA-binding proteins discussed have been prepared to provide absorption spectra that can be easily resolved from that of DNA. The other DNA-binding proteins all appear to have wild-type function. However, to date, there is not enough data available to make similar generalizations about eukaryotic proteins.