Optimized Zebrafish Apolipoprotein A-I Expression and Purification for Nabbs Assembly

G protein-coupled receptors (GPCRs) are heptahelical transmembrane proteins that are highly susceptible to their lipid environment. Reconstitution of GPCRs into detergent micelles or liposomes allows researchers to control the lipid environment. Yet, micelle and liposome preparations are in homogeneous and protein incorporation is difficult to control. To address these issues, nanoscale apolipoprotein bound bilayers (NABBs), soluble phospholipid bilayers stabilized by the helical protein apolipoprotein A-I derived from zebra fish (zap1), have been previously devised. Yet, our approach has been limited by the poor expression of zap1 in E. coli. [1] To increase expression of zap1, we designed a synthetic gene (zapN1) with a codon-optimized sequence encoding the same amino acid sequence as zap1. We transformed BL21-Gold (DE3) cells with the zapN1 gene and performed a double-screen of protein expression from single transformed colonies. We show that zapN1 expresses in a shaking culture at higher levels than the native gene and the membrane scaffold protein MSP1D1 from Stephen Sligar's group. We carefully parameterized the induction and temperature values, finding that induction at an OD600 of 0.8 with 1 mM IPTG and 3 hours incubation at 30 °C yields the highest protein yield per ml of culture. We also show that a batch purification procedure of zapN1 using the His60 Ni superflow resin yields highly pure zapN1. The optimized protocol is easy to implement and requires no extensive preparation with fermenter or FPLC purification. The exceptional purity and high yield of the procedure makes it attractive for single-molecule fluorescence assays, such as fluorescence (cross-) correlation spectroscopy and total internal reflection fluorescence microscopy, and structural studies. [1] AM Knepp, A Grunbeck, S Banerjee, TP Sakmar, T Huber (2011) Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands. Biochemistry, 50, 502-511.