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

Wanxia Li Tsai, Jeffrey G. Forbes, Kuan Wang

研究成果: 雜誌貢獻文章同行評審

3 引文 斯高帕斯(Scopus)


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.

頁(從 - 到)187-199
期刊Protein Expression and Purification
出版狀態已發佈 - 10月 1 2012

ASJC Scopus subject areas

  • 生物技術


深入研究「Engineering of an elastic scaffolding polyprotein based on an SH3-binding intrinsically disordered titin PEVK module」主題。共同形成了獨特的指紋。