SEQUENCE OF 3 CDNAS ENCODING AN ALKALINE MIDGUT TRYPSIN FROM MANDUCA-SEXTA

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 tryp sin, a DNA probe was generated using the polymerase chain reaction (PC R) with template isolated from a midgut cDNA library phage stock, a mi xture of degenerate primers synthesized to code for the highly conserv ed 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 signa l sequence of 17 amino acids, an activation peptide of seven amino aci ds and a mature trypsin of 232 amino acids. The encoded midgut trypsin s contain the highly conserved residues, Asp, His, Ser, involved in ca talysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that ah sequence s are similar to other invertebrate and vertebrate serine proteases, b ut they differ in that two of the three encoded trypsins have an odd n umber of cysteines. Northern analysis localizes the trypsin mRNA to th e 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.