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March 7th, 2025
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Florida State University
molecular biology
biorxiv

A Robust Expression and Purification Protocol for the Production of the La Domain of Human LARP6

Gordon, B. H.Open in Google Scholar•Blackford, N.Open in Google Scholar•Silvers, R.Open in Google Scholar

Human La-related protein 6 (HsLARP6) regulates the highly organized biosynthesis of type I procollagen polypeptides and affects proper assembly of procollagen peptides into heterotrimers of type I procollagen. HsLARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop (5'SL) motif found in type I and III collagen messenger RNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic propensity of the La domain of HsLARP6 to aggregate hampers studies probing the molecular basis of biologically- and disease-relevant structure-function relationship, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effect of the protein construct length and RNA binding on protein stability. C terminal truncations greatly impact protein stability, while N-terminal truncations have little to none effect on protein aggregation and RNA binding. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50x longer than the unbound state, under identical conditions. These results provide a significant platform for further studies of the molecular recognition of 5'SL by HsLARP6.

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