1- Progress on folding reporters 2- New protein tagging and detection system

Circular permutant GFP insertion vector (GFPi) IRBS or truncations are non-fluorescent X Standard X-GFP fusion GFP Circular Permutant Insertion Fluorescent iRBS or truncation

Improved evolution protocol START: Wild type Target X Insoluble (1) Shuffle X N C X (5) Recombine Optima 3-5 (1 month) (2) Clone into new folding reporter (3) Pick Brighter Clones (4) Screen for size Single colony PCR 100 m STOP: Screen optima for solubility

New protein tagging and detection system

  GFP microdomain tagging L L X 1-10 X Soluble R 1-10 Misfolded Aggregated 1-10

The large fragment 1-10 was engineered to improve performance and solubility In vitro In vivo +1-10 wt +1-10 opt SR-s11wt 200 400 600 800 1000 100 300 t (min) Fluorescence Evolved 1-10 wt 1-10 (x3)

Small S11 tag was engineered to minimize its effect on passenger folding and solubility HPS-S11 fusions HPS WT OPT S P 6 14 21.5 31 36.5 66.3 97.4

In vitro complementation assay Express X-s11 Sonication Centrifugation T7 X s11 pET 28a soluble insoluble dissolve in 9M Urea KmR + large fragment Fluorescence reading

In vivo complementation assay tet X s11  Clone proteins in fusion with small S11 fragment in a pPROTet vector  Transform in a E. coli BL21 (DE3) strain harboring the large 1-10 fragment on a pET plasmid  Induce sequentially: first the tagged fusion protein, then the large complementary fragment. colE1 PROTet-6xHN SR lac (1-10) pET 28a p15 KmR

In vivo cell fluorescence Sequential Induction→solubility Co-induction→expression 1 9 17 1 9 17 2 10 18 2 10 18 3 11 3 11 4 12 4 12 5 13 5 13 6 14 6 14 7 15 7 15 8 16 8 16

Summary Folding and solubility are two halves of the equation. We are developing toolbox based on GFP: Folding →GFP CPI vector Solubility →split GFP assay Applications of the solubility reporter: Protein quantification, solubility screening in high-throughput structural genomics Advantages: one step protocol ( opposite to Dot-Blot system.. Several steps) Sensitive, non-perturbing Works in vivo and in vitro Genetically encoded, straight-forward to produce 1-10 FOR MORE DETAILS, SEE POSTER…

Acknowledgments Individual Institute Project Funding Geoffrey S. Waldo LANL Directed Evolution TB PI NIH/LDRD Split GFP Stephanie Cabantous LANL Split GFP LDRD/NIH Brian Mark LANL Directed Evolution TB NIH Jean-Denis Pedelacq LANL Directed Evolution TB NIH Yvonne Rogers LANL Directed Evolution TB NIH Cleo Naranjo LANL Directed Evolution TB NIH Thomas C. Terwilliger LANL TB Project PI NIH