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

Abstract

Summary: 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 reprogramming. Interestingly, the addition of charged and hydrophilic amino acids, such as glutamate or lysine stabilizes KLF4, enhancing reprogramming phenotypes. These findings raise awareness that N-terminal modification with 2A peptide-derived proline or additional cloning conventions may affect protein stability within polycistronic constructs. : In this article, Woltjen and colleagues show that protein expression levels in polycistronic reprogramming constructs depend upon the method of 2A peptide linkage. Specifically, the 2A peptide-derived N-terminal proline along with non-native amino acids derived during cloning affect the stability of KLF4. Their results correlate with discrepancies in reprogramming outcomes and reports of cDNA order-effects in other 2A-linked constructs. Keywords: 2A peptide, stoichiometry, KLF4, protein stability, proline/N-end rule, polycistronic cloning, MG132, cycloheximide, iPSCs, reprogramming