Using site-directed mutagenesis to engineer human ribosomal protein variants

Abstract

We used a site-directed mutagenesis (SDM) protocol to create plasmid constructs through substitution and deletion. When the pmTurquoise plasmid containing the coding sequence for human ribosomal protein (RP) S6 was originally synthesized, a single nucleotide was added, shifting the reading frame of the mTurquoise gene out of line with RPS6. SDM was used to delete nucleotide 3619 and restore the fusion protein reading frame. Since we are using these plasmids to study the extraribosomal functions of RPS6, two different plasmids were constructed with this deletion: control (pmTurquoise-N1-RPS6) and one containing a nuclear export signal (pmTurquoise-NES-RPS6). On a pNH-TrxT bacterial expression plasmid containing the coding sequence for human RPS3A, SDM was also used to substitute amino acids at positions previously identified as post-translational glycosylation sites. On this plasmid, the serine at position 59 and the serine at position 154 in the wild-type RPS3A sequence were substituted individually with alanine to disrupt the residue targeted by the modification. Mutagenesis efficiency was estimated at >80% by blue-white colony screening of control reactions, and designed nucleotide changes were confirmed by plasmid DNA sequence analysis. The constructs on the pmTurquoise backbone will allow us to study the activity of RPS6 in the cytoplasm upon transfection into HEK293 cells. The pNH-TrxT-RP plasmids will enable bacterial protein expression and affinity purification of human RPs from soluble and insoluble fractions for subsequent in vitro glycosylation assays.