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).
Knott
J A
JA
Genetics Unit, Glaxo Group Research Limited, Greenford, England.
Sullivan
C A
CA
Weston
A
A
D000123:Acetyltransferases / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001011:Aorta / Q000187:drug effects; D009320:Atrial Natriuretic Factor / Q000302:isolation & purification* / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012995:Solubility; D013917:Thrombin
D000596:Amino Acids; C472955:NPPA protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009320:Atrial Natriuretic Factor; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D013917:Thrombin