Title Recombinant expression and purification of human pcif1, a cap-specific adenosine-n6 methyltransferase /
Translation of Title Recombinant Expression and Purification of Human PCIF1, a Cap-Specific Adenosine-N6 Methyltransferase.
Authors Ali, Yaniv
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Pages 18
Keywords [eng] Recombinant protein expression, Escherichia coli expression system, mRNA modifications, m6Am, cap-specific methylation, PCIF1/CAPAM, chaperone-assisted folding, solubility optimization, Phosphorylated CTD Interacting Factor 1, mRNA Cap Adenosine N6-Methyltransferase
Abstract [eng] RNA modifications have gained increasing attention due to their critical roles in gene regulation and expression. One such modification, N^6,2'-O-dimethyladenosine (m^6Am), significantly enhances mRNA stability and resistance to degradation. This modification is catalyzed by the cap-specific methyltransferase PCIF1. However, the recombinant production of human PCIF1 is challenging because of its structural complexity and tendency to aggregate when expressed in bacterial systems. Therefore, this study aimed to express human PCIF1 recombinantly in Escherichia coli and to develop strategies to achieve soluble production and efficient isolation. The PCIF1 gene was cloned into suitable expression vectors and transformed into E. coli. Initial attempts to express His-tagged PCIF1 (without additional fusion proteins) resulted in poor solubility, with the protein accumulating predominantly in inclusion bodies. To improve protein solubility, fusion tags were employed, leading to enhanced folding and significantly increased soluble protein expression; however, the activity of these tagged constructs was not evaluated. Additionally, co-expression with molecular chaperones (GroEL/GroES) notably improved the solubility of His-tagged PCIF1 (no fusion protein), clearly demonstrating their beneficial role in protein folding. Both tagged and His-tagged versions of PCIF1 were successfully isolated by affinity chromatography; however, additional purification steps are necessary to achieve high purity. In conclusion, this study demonstrates that soluble expression of human PCIF1 in E. coli can be effectively achieved either by using fusion tags or through chaperone-assisted folding. These findings offer promising strategies for future structural and functional studies, as well as biotechnological applications involving this important enzyme.
Dissertation Institution Vilniaus universitetas.
Type Master thesis
Language English
Publication date 2025