Ni2+ Catalyzed Cleavage of TrpLE‐Fused Small Transmembrane Peptides

• Published in: Chembiochem : a European journal of chemical biology Vol. 23; no. 2; pp. e202100514 - n/a
• Main Authors: Tang, Meng, Cao, Ruiyu, Du, Lingyu, Xu, Jikang, Wu, Bin, OuYang, Bo
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley Subscription Services, Inc 19-01-2022
• Subjects: Amino acid sequence; Cleavage; Clustering; Coronaviruses; Cyanogen; Fusion protein; Hydrophobicity; Membrane proteins; Membranes; Nickel; NMR; NMR spectroscopy; Nuclear magnetic resonance; Peptides; Protein purification; Proteins; Reagents; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; single-pass transmembrane proteins; SNAC-tag; Structural analysis; transmembrane domain; TrpLE
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202100514

In addition to a membrane anchor, the transmembrane domain (TMD) of single‐pass transmembrane proteins (SPTMPs) recently has shown essential roles in the cross‐membrane activity or receptor assembly/clustering. However, these small TMD peptides are generally hydrophobic and dynamic, difficult to be expressed and purified. Here, we have integrated the power of TrpLE fusion protein and a sequence‐specific nickel‐assisted cleavage (SNAC)‐tag to produce small TMD peptides in a highly efficient way under mild conditions, which uses Ni2+ as the cleavage reagent, avoiding the usage of toxic cyanogen bromide (CNBr). Furthermore, this method simplifies the downstream protein purification and reconstitution. Two representative TMDs, including the Spike‐TMD from severe acute respiratory syndrome coronavirus 2 (SARS2), were successfully produced with high‐quality nuclear magnetic resonance (NMR) spectra. Therefore, our study provides a more efficient and practical approach for general structural characterization of the small TM proteins.
Sequence‐specific nickel‐assisted cleavage (SNAC) of TrpLE‐fused transmembrane (TM) peptides avoids the usage of toxic cyanogen bromide and produces small TM peptides in a highly efficient way under mild conditions.