Production and Purification of Slow Skeletal Myosin-binding Protein C N-terminal Fragments

Abstract

Myosin binding protein-C (MyBP-C) is a sarcomeric protein that plays an essential role in regulatingmuscle contraction and maintaining structural integrity in striated muscle. It exists as 3 paralogs expressed in separate genes: slow skeletal (sMyBP-C, MYBPC1), fast skeletal (fMyBP-C, MYBPC2), and cardiac MyBP-C (cMyBP-C, MYBPC3). Mutations in the gene encoding sMyBP-C are implicated in debilitating disorders such as distal arthrogryposis type 1 (DA-1), yet the molecular mechanisms underlying this pathology remain poorly understood. Moreover, sMyBP-C is alternatively spliced in many variants, including a short and long form. Little is known about the differences in function of the skeletal paralogs and these splice variants. Interestingly, sMyBP-C long contains a PKA-mediated phosphorylation site in its N-terminal linker that precedes the C1 domain. This is different from the location of the PKA site in cMyBP-C, which resides in M-domain in between C1 and C2 domains. Therefore, it is likely that the mechanisms of skeletal MyBP-C in health and disease are distinct from cMyBP-C. To enable mechanistic studies, this thesis focuses on producing and purifying human N-terminal domain fragments (C1-C2 domains) of s- and f- MyBP-C using an expression and purification protocol originally developed for c-MyBP-C. The initial constructs of sC1-C2 short, sC1-C2 long, and fC1-C2 each contained a cleavable C-terminal His-tag for purification that contained an 11-residue linker of MyBP-C sequence before the tag termed, 11cHT. These constructs were codon-optimized for bacteria, cloned into the pET45b vector, and expressed in E. coli BL21(DE3) cells. Autoinduction media and a reduced growth temperature (25°C) were used to enhance protein solubility and yield. Following lysis and immobilized metal affinity chromatography (IMAC), proteins were successfully purified and subjected to TEVp cleavage and dialysis. SDS-PAGE analysis confirmed high expression and tag removal, with purity levels ranging from 84% to 94%. Final protein yields were robust (~19 mg per 500 mL culture). These results validate the applicability of the cardiac MyBP-C expression strategy to skeletal paralogs, supporting future studies of actin binding, phosphorylation effects, and pathogenic mutations in skeletal MyBP-C. We note that most recently, optimizations of skeletal C1-C2 protein design and production by the Colson lab resulted in using an N-terminal cleavable His-tags and even lower growth temperatures of 18oC. The purified constructs lay the foundation for biophysical assays such as FRET to explore the structural and functional consequences of DA-associated mutations and the broader role of skeletal MyBP-C in muscle physiology. Rationales and considerations for use of strategies, protocols, and reagents used for production and purification of recombinant MyBP-C are described. Predictions of how such studies previously done in the Colson lab using cMyBP-C may be expected for future studies applied to skeletal MyBP-C biology are discussed.