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

2. 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

3. 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

4. New protein tagging
and detection system

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

6. 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)

7. 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

8. 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

9. 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

10. 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

11. 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…

12. 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