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

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

4. ORF A ORF B + ORF B ORF A

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

6. ORF A ORF B + Ligation: ORF B ORF A

7. ORF A ORF B + Ligation: ORF B ORF A

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

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

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

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

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

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

14. Advantages of the FLIP method: • 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. The Protocol: 1) Digest with a restriction enzyme. 2) Heat inactivate the restriction enzyme (60o, 20min) 3) T4-treat 4) Anneal two plasmids (1 min) and transform into E. Coli. LIC-cloning

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

16. Expression of individual P. falc. proteins SDS Lysate 3C, 20ug EQ2995B EQ2997B EQ3005B EQ3009B EQ3061B EQ3063B EQ3071B EQ3075B EQ3089B EQ3093B EQ3107B EQ3027B EQ3041B EQ3047B MW, 0.4ug EQ3023B EQ3043B C1a C2a C6a C8a D3a D5a D12a E1a E3a C1b C2b C6a C8b D3b D5b D12b

17. Co-expression of P. falc. pairs SDS Lysate 3C, 20ug EQ3255B EQ3256B EQ3262B EQ3263B EQ3264B EQ3265B EQ3266B EQ3267B EQ3268B EQ3258B EQ3259B EQ3261B MW, 0.4ug EQ3257B EQ3260B 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

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

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

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