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Expression and purification of a difficult sarcomeric protein: Telethonin.
Protein Expr. Purif. 140, 74-80 (2017)
Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin1-90) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin1-90 assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin1-90. Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Co-expression ; Protein Expression ; Protein Purification ; Telethonin ; Z1z2; Half-life; Z-disc; Titin; Complex; Muscle; Terminus
ISSN (print) / ISBN
1046-5928
e-ISSN
1046-5928
Quellenangaben
Volume: 140,
Pages: 74-80
Publisher
Elsevier
Publishing Place
San Diego
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Structural Biology (STB)