Recombinant expression and characterization of five-domain Kazal-type serine proteinase inhibitor of black tiger shrimp (Penaeus monodon).
Four full-length complementary DNAs of Kazal-Type serine proteinase inhibitors (SPIs) were identified from hemocyte cDNA libraries of the black tiger shrimp Penaeus monodon and recognized as having 4 (SPIPm1) and 5 (SPIPm2, SPIPm3, and SPIPm4) Kazal domains. SPIPm2 encoding a complete 5-Kazal-domain inhibitor was expressed in the Escherichia coli system. Inhibitory activity of crude proteins against various serine proteases was tested using SPI activity gelatin with sodium dodecylsulfate polyacrylamide gel electrophoresis and inhibitory spectrum assays. A 32-kDa recombinant protein (rSPIPm2) showed inhibitory activity against trypsin, chymotrypsin, and subtilisin, but not elastase. Concordantly, inhibitory spectrum assays showed that crude rSPIPm2 strongly inhibited trypsin (89%) and chymotrypsin (70%), but less effectively inhibited subtilisin (8%), and did not inhibit elastase activity. Northern blot analysis of hemocyte total RNA showed 2 SPI transcripts of 1.6 and 1.7 kb in size. Tissue-specific expression using reverse transcriptase polymerase chain reaction suggests that SPIPm2 is exclusively expressed in hemocytes of P. monodon.
Jarasrassamee
Boonyarin
B
Shrimp Molecular Biology and Genomics Laboratory, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Supungul
Premruethai
P
Panyim
Sakol
S
Klinbunga
Sirawut
S
Rimphanichayakit
Vichien
V
Tassanakajon
Anchalee
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002918:Chymotrypsin / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D033561:Penaeidae / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000378:metabolism*; D020860:Subtilisin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D020860:Subtilisin