Peterson A M, Barillas-Mury C V, Wells M A
Department of Biochemistry, University of Arizona, Tucson 85721.
Insect Biochem Mol Biol. 1994 May;24(5):463-71. doi: 10.1016/0965-1748(94)90041-8.
We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta.
我们从烟草天蛾的中肠中纯化了胰蛋白酶，并证明其最适碱性pH值为10.5。为了克隆中肠胰蛋白酶，使用聚合酶链反应（PCR）生成了一个DNA探针，模板来自中肠cDNA文库噬菌体原种，合成了一组简并引物，用于编码胰蛋白酶中活性位点丝氨酸周围的高度保守区域，以及T7测序引物。分离出了三种不同的胰蛋白酶cDNA，每种都编码一个由256个氨基酸组成的前原酶，其中有一个17个氨基酸的假定信号序列、一个7个氨基酸的激活肽和一个232个氨基酸的成熟胰蛋白酶。编码的中肠胰蛋白酶包含丝氨酸蛋白酶催化过程中涉及的高度保守残基Asp、His、Ser，以及定义胰蛋白酶特异性口袋的残基。序列比较表明，所有序列都与其他无脊椎动物和脊椎动物的丝氨酸蛋白酶相似，但不同的是，三个编码的胰蛋白酶中有两个的半胱氨酸数量为奇数。Northern分析将胰蛋白酶mRNA定位到中肠中间三分之一处。这三个cDNA编码了大量的精氨酸（19、20和21个），这些精氨酸可能通过保持质子化状态，在烟草天蛾的碱性中肠中稳定胰蛋白酶。
