Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms

Abstract

The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to > 2 mg/L using a tandem approach of an N-terminal His6-SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein.

Maternal Larp6 controls oocyte development, chorion formation and elevation

“…Type 1 collagen is abundant, forms fibrils and is mainly distributed in bone, dermis, tendon, ligament and cornea (Gelse et al, 2003). In zebrafish, three type 1 collagen genes exist, col1a1a, col1a1b and col1a2, which code for α1(I), α3(I) and α2(I) chains, respectively, and all retain the conserved Larp6-binding element in their 5′ UTR (Cai et al, 2010;Castro et al, 2017). The three mRNAs are expressed in epidermis, fin, muscles, osteoblasts and fibroblast of the tendons and ligaments (Gistelinck et al, 2016;Thisse and Thisse, 2004).…”

Characterization of sequence specific binding of LARP6 to the 5’ stem-loop of type I collagen mRNAs and implications for rational design of antifibrotic drugs

“…The involvement of two protein domains and five nucleotides of RNA suggested complex interactions between the 5'SL and LARP6. The Kd of 5'SL/LARP6 interaction was measured to be 0.4 nM using fluorescence polarization [10], 12 nM using gel mobility shift assay [12] and 40 nM using ITC [11].…”