Knott J A, Sullivan C A, Weston A
Genetics Unit, Glaxo Group Research Limited, Greenford, England.
Eur J Biochem. 1988 Jun 1;174(2):405-10. doi: 10.1111/j.1432-1033.1988.tb14112.x.
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).
人心房利钠因子[ANF(1 - 28)]已从大肠杆菌中产生的融合蛋白中分离出来。ANF(1 - 28)通过对人凝血酶消化或2-(2 - 硝基苯磺酰基)-3 - 甲基-3'-溴吲哚(BNPS - 粪臭素)的化学作用敏感的独特切割序列，与天然存在的大肠杆菌蛋白氯霉素乙酰转移酶相连。连接序列分别是甘氨酸-缬氨酸-精氨酸-甘氨酸-脯氨酸-精氨酸和色氨酸。释放出的ANF通过反相高效液相色谱法进行纯化。优化的切割条件从具有凝血酶敏感连接子的融合蛋白中释放出5 - 10%(质量)的ANF(1 - 28)最大产量，而在BNPS - 粪臭素处理后，从具有色氨酸连接子的融合蛋白中观察到2 - 5%(质量)的产量。纯化的切割产物具有生物活性，且显示为由完整的ANF(1 - 28)组成。快原子轰击质谱法确认[MH]+为3079 m/z，与ANF(1 - 28)一致。
