Kadaré G, Rozanov M, Haenni A L
Institut Jacques Monod, Paris, France.
J Gen Virol. 1995 Nov;76 ( Pt 11):2853-7. doi: 10.1099/0022-1317-76-11-2853.
The large non-structural polyprotein (206 kDa) of turnip yellow mosaic tymovirus (TYMV) undergoes auto-cleavage, producing N- and C-terminal proteins. Here we show that the viral proteinase responsible for this event is active when produced in Escherichia coli, as monitored in Western blots by examining the generation of the C-terminal cleavage product after induction by IPTG. The outer boundaries and critical amino acids of the proteinase domain were characterized by deletion analysis and site-directed mutagenesis. A miniproteinase of 273 residues resulting from combined N- and C-terminal deletions still performed efficient cleavage. Sequence analysis of the bacterially-purified C-terminal cleavage product indicated that cleavage occurs between Ala1259 and Thr1260 of the non-structural protein.
芜菁黄花叶芜菁黄花叶病毒（TYMV）的大型非结构多聚蛋白（206 kDa）会进行自我切割，产生N端和C端蛋白。在此我们表明，负责此过程的病毒蛋白酶在大肠杆菌中表达时具有活性，通过IPTG诱导后，在蛋白质免疫印迹中检测C端切割产物的产生来进行监测。通过缺失分析和定点诱变对蛋白酶结构域的外部边界和关键氨基酸进行了表征。由N端和C端联合缺失产生的273个残基的微型蛋白酶仍能高效切割。对细菌纯化的C端切割产物的序列分析表明，切割发生在非结构蛋白的Ala1259和Thr1260之间。
