Design of a yeast SUMO tag to eliminate internal translation initiation.

Abstract

After overexpression in a suitable host, recombinant protein purification often relies on affinity (e.g., poly-histidine) and solubility-enhancing (e.g., small ubiquitin-like-modifier [SUMO]) tags. Following purification, these tags are removed to avoid their interference with target protein structure and function. The wide use of N-terminal His₆-SUMO fusions is partly due to efficient cleavage of the SUMO tag's C-terminal Gly-Gly motif by the Ulp1 SUMO protease and generation of the native N-terminus of the target protein. While adopting this system to purify the Salmonella homodimeric FraB deglycase, we discovered that Shine-Dalgarno (SD) sequences in the eukaryotic SUMO tag resulted in truncated proteins. This finding has precedents for synthesis of partial proteins in Escherichia coli from cryptic ribosome-binding sites within eukaryotic coding sequences. The SUMO open reading frame has two "GGNGGN" motifs that resemble SD sequences, one of which encodes the Gly-Gly motif required for Ulp1 cleavage. By mutating these SD sequences, we generated SUMO^NIT (no internal translation), a variant that eliminated production of the truncated proteins without affecting the levels of full-length His₆-SUMO-FraB or Ulp1 cleavage. SUMO^NIT should be part of the toolkit for enhancing SUMO fusion protein yield, purity, and homogeneity (especially for homo-oligomers). Moreover, we showcase the value of native mass spectrometry in revealing the complications that arise from generation of truncated proteins, as well as oxidation events and protease inhibitor adducts, which are indiscernible by commonly employed lower resolution methods.