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Replacement of phenylalanine residues in a protein with p-fluoro-L-phenylalanine (F-Phe) can be implemented for the structural analysis of the protein by19F NMR8, 9. However, as with selenomethionine substitution, F-Phe substitution is cytotoxic. We followed the expression of the CspA in a manner analogous to that demonstrated for selenomethionine EnvZB. CspA was expressed with pColdI(SP4)cspA for 21 hr with and without the addition of F-Phe (FIG. 4A). The addition of F-Phe did not adversely affect CspA production in the SPP system. We then affinity purified the 86-residue CspA product and used mass spectroscopy to determine the extent of F-Phe substitution (FIGS. 4B,C). Although the endogenous Phe biosynthesis was not inhibited in the present SPP system, substantial incorporation of F-Phe (approximately 60 to 70% of total Phe residues) was observed, indicating that toxic F-Phe can be efficiently incorporated into a protein without affecting its yield.

1. customWe then affinity purified the 86-residue CspA product
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