OPTIMIZATION OF SOLUBILITY ENHANCEMENT, SOLUBILIZATION, AND PURIFICATION OF RECOMBINANT AVIAN MYELOBLASTOSIS VIRUS REVERSE TRANSCRIPTASE ?-SUBUNIT

• Main Author: Kameliani, Desti
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/56578
• Institution: Institut Teknologi Bandung

Reverse transcriptase (RTase) is an enzyme that has the activity as DNA polymerase from both DNA and RNA templates, and specific RNase H activity on the RNA strand of the RNA:DNA hybrid. RTase from Avian myeloblastosis virus (AMV) is a heterodimer protein consisting of ?-subunit (63 kDa) and ?-subunit (95 kDa), with better temperature stability and higher potency than RTase from Moloney murine leukemia virus (MMLV). The AMV RTase ?-subunit mutant V238R/L388R/D450A was produced in a previous study in Escherichia coli as an inclusion body. Therefore, this study aims to increase AMV RTase ?-subunit solubility using various E. coli BL21(DE3) strains and chaperones co-expression, as well as to optimize the solubilization and purification processes. Overproduction was carried out using E. coli BL21(DE3), E. coli BL21-Gold(DE3), and E. coli BL21-Codonplus (DE3)- RIPL. Overproduction was also carried out using GroES/EL-TF, DnaKJE, and DnaKJE-GroES/EL chaperone systems in E. coli BL21 (DE3). Overproduction condition were optimized by various temperature at 37?, room temperature (26-28?), and 15?. The results of overproduction with various strains of E. coli BL21(DE3), chaperones co-expression, and lower temperature were not able to increase AMV RTase ?-subunit solubility. AMV RTase ?-subunit inclusion body from E. coli BL21-Gold(DE3) was then solubilized using 8 M urea and renatured using Larginine 0.1 and 0.25 M. The RT-qPCR results showed that AMV RTase ?-subunit had lower activity than commercially AMV RTase kit. These results suggest that the protein refolding was not successful in restoring the correct folding AMV RTase to form functional protein conformation.