[Gene expression, purification and functional analysis of LiPrmA]

Area of Science:

• Molecular Biology
• Microbiology
• Biochemistry

Background:

• Leptospira interrogans is a bacterial pathogen.
• Ribosomal protein L11 methyltransferase (PrmA) plays a role in bacterial ribosome function.
• The specific function of liPrmA in L. interrogans was not well understood.

Purpose of the Study:

• To clone, express, and purify the ribosomal protein L11 methyltransferase (liPrmA) from Leptospira interrogans.
• To characterize the enzymatic activity of the purified liPrmA.
• To investigate the potential role of liPrmA in L. interrogans.

Main Methods:

• Polymerase Chain Reaction (PCR) was used to amplify the liprmA gene.
• The gene was cloned into an expression plasmid (pET22b-/liprmA) in E. coli.
• Recombinant liPrmA-6xHis fusion protein was expressed in E. coli BL21 and purified using Ni-NTA His Bind chromatography.
• Amino acid homology analysis and in vitro methylation assays were performed.

Main Results:

• The full-length liprmA gene was successfully cloned and expressed.
• High yield (40 mg/L) of soluble recombinant liPrmA-6xHis fusion protein was obtained.
• Purified liPrmA demonstrated significant amino acid identity with other PrmA proteins, particularly in the catalytic and AdoMet binding domains.
• The purified liPrmA exhibited catalytic activity, methylating Leptospira interrogans ribosomal protein L11 in the presence of S-adenosyl-methionine (AdoMet).

Conclusions:

• The study successfully produced and purified active liPrmA from Leptospira interrogans.
• The findings confirm liPrmA's role as a methyltransferase for ribosomal protein L11.
• This research provides a foundation for understanding the function and potential therapeutic targeting of liPrmA in L. interrogans.