EXPRESSION ANALYSIS OF tuf GENE FROM Manihot esculenta Crantz (CASSAVA) ON PROKARYOT SYSTEM AS A MOLECULAR CHAPERON

• Main Author: Badriyah, Lailatul
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34984
• Institution: Institut Teknologi Bandung

One of the proteins involved in the mechanism of resistance to high temperatures in prokaryotes is the elongation factor-Tu (EF-Tu) encoded by the tuf gene. Research on corn and wheat showed that EF-Tu protein from chloroplasts also acts as a molecular chaperon that helps refolding of proteins denatured due to heat stress. Chloroplast EFTu proteins are conserved in eukaryotic plants, so it is suggested that EF-Tu from other plants also act as a molecular chaperon. Cassava tuf gene that encode chloroplast EF-Tu protein was used in this study. Cassava tuf genes were cloned in pGEM -T Easy and constructed into the pET-32b via the method of cut and ligate. Recombinant plasmid pET-32b then was used to transform Escherichia coli BL21 (DE3). The next stage was viability test at 37°C, 50°C, 52°C, 53°C, 54°C and 55°C with IPTG induction and protein analysis using SDS-PAGE. Sequencing results by Macrogen showed that cloned genes sequences have about 99% similarity with the tuf genes of cassava. The result of viability test showed that E. coli BL21 DE3 transformant can survive after heat-shock treatment at 54°C for 2 hours. SDS-PAGE results showed a protein of approximately 40 kDa in crude extract protein of bacterial transformant, while in control bacteria, that protein was not found. That band was predicted to be the protein expressed by tuf gene. Analysis of protein motifs using tools pSITE Softberry indicated a protein kinase C phosphorylation site and casein kinase II phosphorylation site that are involved in exchanging GDP into GTP. Based on the above results, it can be indicated that EF-Tu protein from cassava acts as a molecular chaperon.