Bioinformatic approach for Generation of a Fusion Protein of t- snare Snap- 23 Containing A Tronsmembrane Domain

Abstract

Two trans-membrane human proteins namely MDR3 and CLP24 were selected by using online protein databases; UniProtKB, Interpro and NCBI. These proteins have multispanning

membrane topology that belonged to non-raft regions of the membranes. Membrane topology was predicted using an online tool TMMOD. Similarly, online tools; CSS-Palm, NMT-MYR, GPI-SOM were used to predict the post translational modifications like Palmitoylation, myristoylation and GPI-anchor signals respectively in the various polypeptide segments of the selected proteins. These proteins were then narrowed down to specific polypeptide segments that did not show post-translational modifications. The selected polypeptide sequences are MDR3 (AA, 1-145), MDR3 (AA, 41-145) and CLP-24 (AA, 134-195). Gene expression profile of these proteins were studied in various human normal and cancer cell lines using ‘GeneNote’ and found that MDR3 and CLP4 were expressed in higher level in liver and lung cells respectively. Insilico fusion proteins hSNAP-23cys - -MDR3, 1-145; hSNAP-23cys - -MDR3, 41-145 and hSNAP-23cys - -CLP24, 134-195 were generated using restriction mapper tool and then the fusion proteins were checked for membrane topology and absence of any posttranslational modifications in them RNA was isolated HeLa and A549 cells using Trizol reagent and then its quality was checked using Nanodrop. The Amount of RNA recovered was 8.13 ug/million HeLa cells and 5.76 ug/million A549 cells. These RNA were reverse transcribed intocDNA and then complementary DNA was tested by agarose gel electrophoresis and performing PCR for human GAPDH gene. Specific primers and QIAGEN Onestep PCR kit were used to isolate the DNA fragments for MDR3 (AA, 1-145) and CLP24 (AA, 134-195). These DNA amplicon could be further used to generate fusion protein of SNAP-23 whose expression could then be studied in specific host cell lines after successful transfection. Though the study of expression of the fusion protein remains to be tested in cell lines, bioinformatics studies showed that these fusion proteins would not be signaled to raft region of membrane and hence may prevent the exocytosis process mediated by SNAP-23 proteins in raft regions. This study has helped in exploring the role of cysteine molecules and their palmitoylation in targeting and anchoring of t-SNARE SNAP-23 molecules to the lipid raft region of the membrane by bioinformatic approach where membrane fusion is supposed to occur by SNARE complex.

Key Words: MDR3, CLP24, UniProt, InterPro, NCBI, TMMOD, GeneNote, SNAP-23, SNARE