N-Terminal Amino Acids Determine KLF4 Protein Stability in 2A Peptide-Linked Polycistronic Reprogramming Constructs.

ABSTRACT: A common strategy for multi-protein expression is to link genes by self-cleaving 2A peptide sequences. Yet, little is known how the 2A peptide-derived N-terminal proline or adjacent non-native residues introduced during cDNA cloning affects protein stoichiometry. Polycistronic reprogramming constructs with altered KLF4 protein stoichiometry can influence induced pluripotent stem cell (iPSC) generation. We studied the impact of N-terminal 2A peptide-adjacent residues on the protein stability of two KLF4 isoforms, and assayed their capacity to generate iPSCs. Here, we show that the N-terminal proline remnant of the 2A peptide, alone or in combination with leucine, introduced during polycistronic cloning, destabilizes KLF4 resulting in increased protein degradation, which hinders reprogrammin

SUBMITTER: Reinhardt A 

PROVIDER: S-EPMC7066363 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature