Engineering of an elastic scaffolding polyprotein based on an SH3-binding intrinsically disordered titin PEVK module

• Published in: Protein expression and purification 2012-10, Vol.85 (2), p.187-199
• Main Authors: Tsai, Wanxia Li, Forbes, Jeffrey G., Wang, Kuan
• Format: Article
• Language: English
• Subjects: Baculoviridae - genetics; Blotting, Western; Connectin; Escherichia coli - genetics; Functionalized elastomer; Glucose - metabolism; Humans; Models, Molecular; Muscle Proteins - chemistry; Muscle Proteins - genetics; Muscle Proteins - metabolism; Pichia - genetics; Plasmids; Polyproteins - chemistry; Polyproteins - genetics; Polyproteins - metabolism; Protein Engineering; Protein Kinases - chemistry; Protein Kinases - genetics; Protein Kinases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; src Homology Domains; Tandem repeat biopolymer; Tandem Repeat Sequences
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2012.08.003

► Successfully engineered titin PEVK polyproteins for nanomechanical measurements. ► Investigated systematically expression hosts for proteins with tandem repeats. ► Expressed and purified gram quantities of secreted polyproteins in a yeast system. ► Facilitated studies of the role of force in titin’s function. Titin is a large elastic protein found in muscle that maintains the elasticity and structural integrity of the sarcomere. The PEVK region of titin is intrinsically disordered, highly elastic and serves as a hub to bind signaling proteins. Systematic investigation of the structure and affinity profile of the PEVK region will provide important information about the functions of titin. Since PEVK is highly heterogeneous due to extensive differential splicing from more than one hundred exons, we engineered and expressed polyproteins that consist of a defined number of identical single exon modules. These customized polyproteins reduce heterogeneity, amplify interactions of less dominant modules, and most importantly, provide tags for atomic force microscopy and allow more readily interpretable data from single-molecule techniques. Expression and purification of recombinant polyprotein with repeat regions presented many technical challenges: recombination events in tandem repeats of identical DNA sequences exacerbated by high GC content, toxicity of polymer plasmid and expressed protein to the bacteria; early truncation of proteins expressed with different numbers of modules; and extreme sensitivity to proteolysis. We have investigated a number of in vitro and in vivo bacterial and yeast expression systems, as well as baculoviral systems as potential solutions to these problems. We successfully expressed and purified in gram quantities a polyprotein derived from human titin exon 172 using Pichia pastoris yeast. This study provides valuable insights into the technical challenges regarding the engineering and purification of a tandem repeat sequence of an intrinsically disordered biopolymer.