Production of thermostable recombinant Cas9 enzyme from Geobacillus kaustophilus TBUI01 in Escherichia coli BL21 with ammonium sulfate precipitation purification method

Michella Anastacia Sinaga, Kenny Lischer

Research output: Contribution to journalConference articlepeer-review

Abstract

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.

Original languageEnglish
Article number070009
JournalAIP Conference Proceedings
Volume3215
Issue number1
DOIs
Publication statusPublished - 25 Nov 2024
Event18th International Conference on Quality in Research, QiR 2023 - Bali, Indonesia
Duration: 23 Oct 202325 Oct 2023

Fingerprint

Dive into the research topics of 'Production of thermostable recombinant Cas9 enzyme from Geobacillus kaustophilus TBUI01 in Escherichia coli BL21 with ammonium sulfate precipitation purification method'. Together they form a unique fingerprint.

Cite this