TY - JOUR
T1 - Production of thermostable recombinant Cas9 enzyme from Geobacillus kaustophilus TBUI01 in Escherichia coli BL21 with ammonium sulfate precipitation purification method
AU - Sinaga, Michella Anastacia
AU - Lischer, Kenny
N1 - Publisher Copyright:
© 2024 Author(s).
PY - 2024/11/25
Y1 - 2024/11/25
N2 - The Cas9 enzyme is part of CRISPR-Cas9 which acts as an endonuclease to cut DNA/RNA in specific sequences. Cas9 is useful in the fields of health, food, and industry. However, many industries operate at high temperatures so that Cas9 enzymes generally cannot be used and a thermostable Cas9 enzyme is needed. Nevertheless, there has not been much research on the type of thermostable Cas9 enzyme. Therefore, knowing the presence of Cas9 in the local isolate Geobacillus kaustophilus TBUI01, Cas9 enzyme production was carried out using recombinant techniques on Escherichia coli BL21. The Cas9 enzyme was then heated to remove all mesophilic protein from Escherichia coli BL21 at 50°C, 60°C and 70°C. Then it was purified by ammonium sulfate precipitation technique with 20%, 50% and 80% saturation. Until now, there has been no research on thermostable Cas9 using ammonium sulfate precipitation as the only purification technique. This technique is done because it is economical, fast, and easy to do. The result showed that a heating temperature of 60°C is the optimal temperature for degrading mesophilic proteins without degrading Cas9 enzymes. Optimal ammonium sulfate precipitation is carried out at 50% fractionation because it can precipitate the Cas9 enzyme. However, there are still other proteins that have been successfully precipitated so that the precipitation of ammonium sulfate can be used as an initial purification step to concentrate protein.
AB - The Cas9 enzyme is part of CRISPR-Cas9 which acts as an endonuclease to cut DNA/RNA in specific sequences. Cas9 is useful in the fields of health, food, and industry. However, many industries operate at high temperatures so that Cas9 enzymes generally cannot be used and a thermostable Cas9 enzyme is needed. Nevertheless, there has not been much research on the type of thermostable Cas9 enzyme. Therefore, knowing the presence of Cas9 in the local isolate Geobacillus kaustophilus TBUI01, Cas9 enzyme production was carried out using recombinant techniques on Escherichia coli BL21. The Cas9 enzyme was then heated to remove all mesophilic protein from Escherichia coli BL21 at 50°C, 60°C and 70°C. Then it was purified by ammonium sulfate precipitation technique with 20%, 50% and 80% saturation. Until now, there has been no research on thermostable Cas9 using ammonium sulfate precipitation as the only purification technique. This technique is done because it is economical, fast, and easy to do. The result showed that a heating temperature of 60°C is the optimal temperature for degrading mesophilic proteins without degrading Cas9 enzymes. Optimal ammonium sulfate precipitation is carried out at 50% fractionation because it can precipitate the Cas9 enzyme. However, there are still other proteins that have been successfully precipitated so that the precipitation of ammonium sulfate can be used as an initial purification step to concentrate protein.
UR - http://www.scopus.com/inward/record.url?scp=85212214784&partnerID=8YFLogxK
U2 - 10.1063/5.0239767
DO - 10.1063/5.0239767
M3 - Conference article
AN - SCOPUS:85212214784
SN - 0094-243X
VL - 3215
JO - AIP Conference Proceedings
JF - AIP Conference Proceedings
IS - 1
M1 - 070009
T2 - 18th International Conference on Quality in Research, QiR 2023
Y2 - 23 October 2023 through 25 October 2023
ER -