Structural Genomics of Parasitic Protozoans ( Trypanosoma, Leishmania ) (SGPP East)

1. Structural Genomics of Parasitic Protozoans(Trypanosoma, Leishmania)(SGPP East) Protein Expression at the University of Rochester Mark Dumont, Elizabeth Grayhack. Eric Phizicky

2. Ligation-Independent Cloning(Uses PCR-vector overlap sequences missing one type of nucleotide) Restriction sites ORF Digest PCR, purify Mix ORF and vector ( 5 min. no ligase) Transform E. coli T4 DNA pol. 3’exo + (i.e.) dGTP T4 DNA pol. 3’exo + (i.e.) dCTP

3. Technical issues with LIC 1. Tm of overhangs 2. Removal of dNTPs from PCR products 3. Complete cleavage of vector

4. Advantages of Ligation Independent Cloning 1. Speed: ~ 1 hr T4 polymerase treatment and 5 min annealing 2. Low Background: Reported to be <1% 3. Directional 4. Minimal introduction of extra amino acids in fusions: Requires a run of 14 bases lacking one of the four nucleotides at each end 5. Only requires short PCR primers: (ORF-specific sequences + 14 nucleotides) 6. Low expense: Requires only T4 DNA polymerase, no proprietary reagents.

5. Ligation Independent Cloning Selected References: Aslanidis C, de Jong PJ, Schmitz G (1994) Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products. PCR Methods Appl. 4:172-7. Haun RS, Serventi IM, Moss J. (1992) Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. Biotechniques 13:515-8. Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res.18:6069-74. Novagen Product Information

6. ORF Expression Vectors for Protozoan Soluble Protein Expression PmlI NdeI + Pml1 NdeI Features: Ligation-Independent Cloning-compatible N-terminal non-cleavable His6 in all vectors ATG-His6-ATG……..……....TAA T7 terminator Ribosome binding site ORF T7 promoter PmlI NdeI

7. Expression of a test gene following ligation-independent cloning and transformation into BL21-DE3 (Coomassie stain) Vector + Insert (Independent isolates) TPT1- 8 g Vector Tpt1p

8. Pichia Pastoris for “High Throughput” Membrane Protein Expression 1. “Lower” eukaryote- post-translational modifications 2. Rapid cloning 3. Inexpensive 4. High density cultures leading to high yields per liter 5. Previous history for membrane protein expression

9. Pichia pre-pro-α-factor signal E. coli pelB signal Pichia no signal E. coli no signal Cloning Strategy for Membrane Protein Expression Single PCR product Use ligation independent cloning to insert a single PCR-product into two E. coli vectors and two Pichia vectors

10. Expression Vectors for Protozoan Membrane Protein Expression Current Features: 1. C-terminal non-cleavable His6 (+ c-myc in Pichia) 2. Vectors with and without cleavable N-terminal secretion signals: pre-pro-α-factor followed by kex2p cleavage site in Pichia pelB followed by signal peptidase site in E. coli 3. Favorable translation context in Pichia. Optimal ribosome binding site in E.coli 4. Codons of fusion regions optimized for Pichia and E. coli 5. Zeocin selection for Pichia vector: selectable in E.coli and yeast, allows selection for multicopy integrants in Pichia. Future: 1. Multiple Affinity Tags, protease sites (rhinovirus 3C (Prescision)) 2. Fluorescent expression screening

11. Pichia LIC vectors with C-terminal Tags (Derived from pPicZα from Invitrogen) • Linearize for LIC by cutting at two BsmB1 sites (cuts outside recognition site) • Additional PmlI site between BsmB1 sites to reduce background due to uncut vector • Linearize at BglII and BamHI for integration into Pichia α without signal peptide with pre-pro-α factor signal peptide

12. Technical Assistance: Wayne Bowers Nadia Fedoriw Craig Menges Pichia consultant: Ina Urbatsch Wim Hol

13. E. coli LIC vectors with C-terminal Tags (Derived from pET22b+ from Novagen) • Linearize by cutting at two sites with Bsm1 • (cuts outside recognition site) • Additional PmlI site between BsmI sites to • reduce background due to uncut vector with pelB signal peptide without signal peptide