1. Membrane Protein Production: 8/06/04 One target shipped: Lmaj000191: Mitochondrial ADP/ATP translocator in 2% octyl glucoside (predominant lower MW band). Other preps: Lmaj000776: (lost due to OG issues, see below) Lmaj001197: (oligomer on gel filtration) Detergent Issues: Protein instability in high octyl glucoside: Lmaj000766, one of our most stable ORFs appears to be rapidly completely degraded in octylglucoside (OG) above the CMC, but not below the CMC. Possible explanations: 1) co-purifying OG-activated protease; 2) OG-activated contamination in 3C protease; 3) oxidative damage via high OG. Successful preparations with nonyl glucoside and undecyl maltoside Limited availbility of Fos-choline 16 Lipid Issues: Extra blobs at bottom of gels are lipid Significant amounts of lipid are carried through purifications (detergent dependent) Added synthetic lipid (DOPC) does not significantly affect purification 3C protease supply for on-column cleavage: One-step-purified 3C works well for on-column cleavage (subject to OG issues, see above). Advantages of small membrane ORFs: LIC cloning of 7 smallest of 800 ORFs from Liz Worthey (truncated and full-length forms) >5/7 give detectable bands by Coomassie staining!

2. Pfal Protein expression in Tetrahymena Primers ordered for soluble protein expression: Soluble, expressed proteins: all have known inhibitors, none have been solved to date. PfAl006677WESPF14_0020Choline kinase PfAl008572WESPFE1430ccyclophilin-like PfAl005921WESPF11_0145Glyoxalase I PfAl005676WESPF10_0289Adensosine deaminase Proteins that expressed but were not soluble (from bacculo list) Pfal000348AAAPf358_t00001protein phosphatase-1 regulatory subunit 7 alpha2-related Pfal005915AAAPF11_0139protein tyrosine phosphatase, putative Pfal005766AAAPF10_0379phospholipase, putative Pfal008186AAAPFD0725carsenical pump-driving ATPase, putative Proteins that did not express (from synthetic gene list) PfAl006912AAAgi|23497305|gb|AE014820.1| exosome component PfAl007889AAAgi|23957744|ref|NP_473209.2|possible dual specificity phosphatase (Note: NNNN…) PfAl006547AAAgi|23619395|ref|NP_705357.1|Topoisomerase III (Note: NNNKKK…) PfAl007173AAAgi|23509739|ref|NP_702406.1|Probable peptidase (Note: NNNKKK…) PfAl005232AAAgi|23612659|ref|NP_704220.1| methyl transferase (Note: NNNKKK…)

3. Primers being ordered for membrane and secreted protein expression in Tetrahymena: LocusFunction/Target rationaleAAs MAL7P1.27pfcrt (choroquine resist) 424 PFE1150wmdr1 (drug resist)1419 PFE1265wGPCR? 467 Pf13_0248pf47 antigen 439 PFB0405s230 antigen3135 AAF63684.1pfs25 antigen 217 AAT00624.1pfs28 antigen 218 PF11_0486MAEBL eryth binding antigen2055 PFA0125cEbl1 erythrocyte binding antigen1567 PFL1315wpfkch1 K+channel1979 PFI0955wput. Hexose transporter 476 PFA0310cCa+ATPase (atemisinin target)1228