The isolation and characterisation of human atrial natriuretic factor produced as a fusion protein in Escherichia coli

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Abstract
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]4 of 3079 m/z, consistent with ANF(1–28).