FIG. 5.

Discrimination of TEVp variants exhibiting different in vivo solubility and isolation of the most soluble one from a large background of a less-soluble TEVp variant. (A) Fluorescence intensity of DH5α/pMal-TEV2/pGFP-ssrA^NY, DH5α/pTEV/pGFP-subG-ssrA^NY, and DH5α/pMal-TEV2/pGFP-subG-ssrA^NY cells, 2.5 h after induction (0.1 mM IPTG, 0.2% arabinose), are shown in red, blue and green, respectively. The plasmid pTEV encodes a TEVp variant, referred to as iTEVp, with poor in vivo solubility due to expression without the solubility-enhancing MBP moiety. On the contrary, pMal-TEV2 encodes TEVp as a transient MBP fusion, which improves the protease's in vivo solubility. (B) DH5α cells coexpressing TEVp and GFP-subG-ssrA^NY were mixed with cells coexpressing iTEVp and GFP-subG-ssrA^NY at a ratio of 1:100,000 (blue). The cell mixture was then subjected to two rounds of flow cytometry analysis and cell sorting to preferentially collect cells expressing the more soluble TEVp variant, and resulted in a 22,000-fold enrichment of cells harboring pMal-TEV2.