The isolation and characterisation of human atrial natriuretic factor produced as a fusion protein in Escherichia coli.
Human atrial natriuretic factor [ANF(1-28)] has been isolated from a fusion protein produced in Escherichia coli. ANF(1-28) was linked to a naturally occurring E. coli protein, chloramphenicol acetyltransferase, via unique cleavage sequences susceptible to either human thrombin digestion, or the chemical action of 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole). The linker sequences were Gly-Val-Arg-Gly-Pro-Arg and Trp respectively. The liberated ANF was purified by reversed-phase HPLC. Optimised cleavage conditions released 5-10% (by mass) of the maximal yield of ANF(1-28) from the fusion protein with the thrombin-susceptible linker, whilst a 2-5% (by mass) yield was observed from the fusion protein with the tryptophan linker after BNPS-skatole treatment. The purified cleavage products were biologically active and shown to comprise intact ANF(1-28). Fast-atom-bombardment mass spectrometry confirmed [MH]+ of 3079 m/z, consistent with ANF(1-28).[1]References
- The isolation and characterisation of human atrial natriuretic factor produced as a fusion protein in Escherichia coli. Knott, J.A., Sullivan, C.A., Weston, A. Eur. J. Biochem. (1988) [Pubmed]
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