Sequencing and molecular cloning of serine peptidases from Tenebrio molitor midgut
Plínio T. Cristofoletti1 ; Fábio K. Tamaki2 and Walter R. Terra2
1: Votorantin Novos Negócios; 2: Instituto de Química IQ-USP
Two serine peptidases were cloned and sequenced from T. molitor midgut cDNA library. These sequences were obtained by random sequencing. One of this serine peptidase is homologous to trypsin (best hit with E value 5e-36 and 57% of amino acids identity with trypsinogen RdoT3 precursor of Rhyzopertha dominica using Blastx search).This sequence codes a protein with the same N-terminal of purified trypsin (IVGGSSISISSVPXQIXLQY) isolated from T. molitor midgut by Tsybina et al., 2005, Biochemistry (Moscow) 70: 300-305. The other serine peptidase is homologous to chymotrypsinogen (best hit with E value 9e-49 and 41% of amino acids identity with Lucilia cuprina chymotrypsinogen using Blastx search). This sequence codes a protein with the same N-terminal of purified chymotrypsin (IISGSAASKGQFPWQ) isolated from T. molitor midgut by Elpidina et al., 2005, Biochimie 87: 771-779. The theorical molecular weight calculated for the mature peptide were 22742.28 kDa for trypsin and 22541.15 kDa for chymotrypsin, and the theorical pI were 6.87 for trypsin and 8.69 for chymotrypsin, which agrees with the chromatografic behavior of these enzymes on ion exchange columns in pH 7.0 (trypsins are not retained on columns, instead of chymotrypsins, that are retained and eluted with linear gradient of NaCl). In both sequences there are the homologous catalytic triad residues, corresponding to the His 44, Asp 88 and Ser 180 for chymotrypsin and His 41, Asp 86 and Ser 182 for trypsin.
One major trypsin activity was purified from T. molitor midgut to homogeneity by a combination of chromatographyc steps. N-terminal microssequencing of this protein resulted in the sequence IVGGSVINDQLVPQNXS, that differs from the isolated clone. Attempts to clone a cDNA coding for this protein are in progress.
Supported by: FAPESP, CNPq.
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