Expression, Purification And Characterization Of Hylaserpin Analogues, Two Trypsin Inhibitors, From Bufo Melanostictus

Bufo melanostictus is mainly distuibuted in Yunnan, Sichuan, Guizhou, Fujian, Jiangxi, as well as Southeast Asia, South Asia, indo-china peninsula and so on. The skin of the B. melanostictus is the main sourch of chanyi, which is used to treat various diseases, although its active compounds remain unclear. Trypsin inhibitors (TI) are found widely in plants, animals and miro-organisms and play various important physiological functions. In this study, two trypsin inhibitors, Hylaserpin analogues1 and Hylaserpin analogue 2, were cloned from skin of Bufo melanostictus. Two oligonucleotide primers,5’ PCR primer F1 (5’-TGYGGNWSNGCNTGYCCN-3’) and 5’ PCR primer F2 (5’-CCNTGYGTNGARGGNTGYGTNTGY-3’), in the sense direction, were designed according to the conserved trypsin inhibitor like cysteine rich domain. Nested PCRs were performed together with 3’ primer R1 in the antisense direction to amplify the 5’ ends of the cDNA. Then two primers, 3’ primer R2 (5’-ATGGACATTCTTCCTTTGGGA-3’) and 3’ primer R3 (5’-AGGACCTATAACATATCC-3’) were designed according to the sequenced nucleotide sequence, together with 5’ PCR Primer Ⅱ A, to amplify the 5’ ends of the cDNA. The nucleotide sequence was analyzed by BLAST and Translate Tool to deduce the amino acid sequence. Two hylaserpin homologue, Hylaserpin analogue 1 and Hylaserpin analogue 2, were deduced from the nucleotide sequences.The open reading frames of Hylaserpin analogue 1 and Hylaserpin analogue 2 were composed of 110 amino acids and 83 amino acids, respectivly. Based on SignalP analysis, Hylaserpin analogue 1 may not contain signal peptide and unlikely to be exposed to the glycosylation machinery, Hylaserpin analogue 2 may contain a signal peptide with 24 amino acids.Recombinant Hylaserpin analogues expressed and purified from Escherichia coli exhibited obvious inhibitory activity against trypsin.The inhibition percentage increased with increasing Hylaserpin analogues concentrations. At a concentration of 2μM, Hylaerpin analogue 1 can inhibite about 36% of trypsin activity within 18s and about 62% at 3 min, Hylaerpin analogue 2 inhibited about 20% of trypsin activity within 18s and 24% at 3 min. At 8 μM, Hylaerpin analogue 1 inhibited nearly 46% of trypsin activity, and about 78% of trypsin activity within 3 min, Hylaserpin analogue 2 inhibited nearly 39% of trypsin activity, and about 47% of trypsin activity within 3 min.In summary, two trypsin inhibitors, Hylaserpin analogues1 and Hylaserpin analogue 2, were isolated from the skin cDNA library of B. melanostictus. Further study of the function of Hylaserpin analogues is required to interpret its role in host immunity and its pharmacological effect.