Audtho M, Valaitis A P, Alzate O, Dean D H
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Appl Environ Microbiol. 1999 Oct;65(10):4601-5. doi: 10.1128/AEM.65.10.4601-4605.1999.
Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.
苏云金芽孢杆菌的Cry2Aa1原毒素（分子量63 kDa）经舞毒蛾幼虫中肠液切割后产生两个主要蛋白片段：一个对昆虫具有高毒性的58 kDa片段和一个无毒性的49 kDa片段。在中肠液中，原毒素在1分钟内被加工成58 kDa毒素，但消化1小时后，58 kDa片段在结构域I内进一步切割，产生抗蛋白酶的49 kDa片段。当给昆虫喂食（125）I标记的毒素时，在体内也发现了58 kDa和无毒的49 kDa片段。N端测序显示，蛋白酶切割位点分别位于活性片段和较小片段的Tyr49和Leu144的C端。为防止中肠加工过程中产生无毒片段，通过在PCR中使用一对互补诱变寡核苷酸，将毒素的Leu144分别替换为Asp（L144D）、Ala（L144A）、Gly（L144G）、His（L144H）或Val（L144V），构建了5种突变蛋白。所有突变蛋白对中肠蛋白酶和胰凝乳蛋白酶都具有高度抗性。除L144H在残基144处被部分切割外，昆虫中肠提取物和胰凝乳蛋白酶对突变蛋白的消化仅产生活性58 kDa片段。
