1. A facile method that allows high-throughput co- expression from plasmids with identical replication origins and antibiotic resistance markers (University of Rochester) SGPP

2. A convenient method to co-express protein pairs (University of Rochester) SGPP

3. Set of expression plasmids: ORF B ORF E Goal: To co-express two proteins using a set of existing plasmids: + 1.Two plasmids, two compatible origins, two selection markers (Amp, Kan) 2.One plasmid: one promoter, polycistronic mRNA 3.One plasmid: two promoters, two ORFs A new plasmid has to be made ? ORF A ORF B ORF C ORF D ORF E

4. ORF A ORF B ORF A +

5. ORF B ORF A + Can it be made? Will it work?

6. ORF A ORF B ORF A + Ligation:

7. ORF A ORF B ORF A + Ligation:

8. 1 2 3 4 1 2 3 4 1 anneals to 4 only 2 anneals to 3 only ORF A ORF B ORF A

9. 1 2 3 4 1 2 3 4 1 anneals to 4 only 2 anneals to 3 only ORF A ORF B ORF A

10. ORF Promoter Expression vector FLIP (61bp) (FLIP version)

11. 1 anneals to 4 only 2 anneals to 3 only

12. ORF B ORF E + ORF B ORF E + ORF B ORF E 1 2 3 4 1 2 4 3 ORF B ORF E 1 2 43 Co-expression of the desired protein pair from the set: Set of expression plasmids: ORF A ORF B ORF C ORF D ORF E

13. empty vector insert 4 insert 3 insert 2 insert 1 2 4 6 8 10 1 0.2 prot.4 12 A Ladder 1 1 + 21 + 31 + 42 + 3 2 + 4 3 + 4 234 Ladder 1 1 + 21 + 31 + 42 + 3 2 + 4 3 + 4 234 D prot.2 prot.1 prot.3 21 14 6 31 45 66 97 116 pLysS C Ladder 1 1 + 21 + 31 + 42 + 3 2 + 4 3 + 4 234 B 4 6 8 10 12 4 6 8 10 12 E3 + E2 abcdefghijk abcdefghijkabcdefghijk 200 Co-expression of all pair combinations for 4 proteins (L. major) (equivalent to 400 Liters of growth): A.Tandem plasmid contains two inserts. B.Tandem plasmid has double size. C.Tandem plasmid propagates in expression cells. D. Protein pairs are expressed: all combinations.

14. It uses an existing set of ORFs in identical plasmids. No primers needed. No PCR used ---> No sequencing needed. No PCR-purification, no gel-purification. No ligation. Octamer restriction enzymes allow to “flip” > 99.1% of possible random protein pairs. No selectable markers or compatible origins to worry about. “FLIP” sequence (61 bp) does not harm, if not used. Advantages of the FLIP method: LIC-cloning The Protocol: 1) Digest with a restriction enzyme. 2) Heat inactivate the restriction enzyme (60 o, 20min) 3) T4-treat 4) Anneal two plasmids (1 min) and transform into E. Coli.

15. P. falciparum pairs Target pairs: obtained in S. Fields lab using yeast two-hybrid screen.

16. MW, 0.4ug 3C, 20ugEQ2995BEQ2997BEQ3005BEQ3009B EQ3041B EQ3027B EQ3023B EQ3043B EQ3047B EQ3061B EQ3063B EQ3071B EQ3075B EQ3093B EQ3089B EQ3107B C1a C2a C6a C8a D3a D5a D12a E1a E3a C1b C2b C6a C8b D3b D5b D12b Expression of individual P. falc. proteins SDS Lysate

17. MW, 0.4ug 3C, 20ugEQ3255BEQ3256B EQ3259B EQ3258B EQ3257B EQ3260B EQ3261B EQ3262B EQ3263B EQ3264B C1a/b C1a/b C2a/b C2a/b C6a/b C6a/b C8a/b C8a/b D3a/b D3a/b D5a/b D5a/b D12a/b D12a/b EQ3265B EQ3267B EQ3266B EQ3268B Co-expression of P. falc. pairs

18. Co-expressed (large scale) and co-purified P. falc. pair E2/E3: EQ3041B – Ubiquitin-conjugating enzyme E2 Ladder E3 + E2 E3E2 21 14 6 45 97 116 31 66 E2 18.2 kDa E3 17.5 kDa 200 EQ3107B – Ubiquitin-protein ligase E3

19. ORF B ORF D + ORF B ORF D "FLIP" ORF A ORF B ORF C ORF D ORF E Goal: To co-express two proteins using a set of existing plasmids: Set of expression plasmids:

20. Erin Quartley Christina de Vries Daniela De Rosa Julie Babulski Yoshiko Kon Sarah Mitchell Elizabeth Grayhack Eric Phizicky Mark Dumont University of Rochester: Stanley Fields Wim Hol Marissa Vignali Doug LaCount Lori Schoenfeld University of Washington (Seattle):