Efficient synchronization of high expression and site-specific biotinylation of the PTBP1 RRM3/4 domain.

Polypyrimidine tract-binding protein 1 (PTBP1), a key regulator of mRNA alternative splicing, has emerged as a promising therapeutic target for neurodegenerative diseases and cancer treatment. Given the critical role of its RRM4 domain's lysine residues in binding to polypyrimidine single-stranded RNA, nonspecific biotinylation could potentially alter PTBP1's activity. Here, we describe a method for the concurrent high-level expression and site-specific biotinylation of the PTBP1 RRM3/4 domain using the pQE80L vector and AVB101 host strain. By optimizing codon usage and GC content, we achieved a maximum expression level of approximately 85 μg/mL in bacterial culture, with over 99 % purity and a 72.4 % biotinylation rate. The recombinant 6His-Avi-PTBP1 demonstrated the ability to emit chemiluminescence when coupled with HRP-streptavidin, to be immunoprecipitated by streptavidin magnetic beads, and to interact specifically with (CU) RNA, exhibiting a dissociation constant (K) of 38 nM as determined by Bio-Layer Interferometry (BLI). Collectively, these results indicate that the recombinant 6His-Avi-PTBP1 is suitable for inhibitor screening and kinetic parameter analysis.