MPP/TMPC OUTSIDE REQUEST FORM Written : 1/25/12 Requestor Reviewed:

1. MPP/TMPC OUTSIDE REQUEST FORM Written: 1/25/12 Requestor Reviewed: Requestor(s):Chuck HoppelExecsReviewed: Institution:Case Western ReserveOutcome: Request Title: Human mitochondrial protein CPT1A, isoform 2

2. - Originally sent 1/25/12 - Forwarded again 2/28/12, suggesting conference call. Dear Dr. Hoppel,We do have somewhat encouraging results on CPT1A. In short, we cloned full-length isoform 2 into a wheat germ cell-free expression vector that adds a C-terminal His tag. As you probably know, isoform 2 has a slightly different C-terminus than isoform 1; we chose to work with it because this form was immediately available to us, but we would appreciate any advice you might have on the isoforms.In initial expression tests using our standard procedures, it expressed well but was only weakly soluble and purified poorly or not at all in small-scale IMAC purification tests.However, when coexpressed with "membrane scaffolding proteins", the protein became highly soluble, though it still purified poorly. These scaffolding proteins are likely forming lipid nanodiscs with lipids found in the wheat germ extract, and are hopefully incorporating CPT1A into the lipid disc.The poor purification may be the result of an inaccessible tag, which might be remedied by moving the tag to the N-terminus or, alternatively, using tags on the scaffolding proteins as a purification handle.In researching CPT1A I was surprised to find that our previous organization, the Center for Eukaryotic Structural Genomics, worked fairly extensively with N-terminal 165 residue-deleted versions of both human and mouse CPT1A. They were expressed in E. coli and we got as far as purifying several hundred  micrograms which checked out by mass spectrometry (both mass and identity by ms/ms sequencing) but was not enough to attempt crystallization. At this point, we would like to obtain advice from you. It may be useful to take a look at the little write-up I've attached, which includes the description (in Requestor Notes) about CPT1A that you sent to Dave Pagliarini in your grant application support letter several years ago. Please correct any misconceptions I may have included and add anything that might be helpful to us, in particular any experience in expressing or purifying the protein or assaying activity. We would also be happy to teleconference with you if that would be useful.Tentative plans are to try some variations on the nanodisc production, possibly try production with liposomes and detergents, and potentially scale up production for which one goal would be to send you some protein for activity assay. We may also resurrect our E. coli effort since we now have some experience with membrane proteins that may produce a more successful outcome (note that the resources of MPP's sister center, the Transmembrane Protein Center, Brian Fox PI, will likely be brought to bear on this target as well).Regards,Dave Aceti