Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity Casim A. Sarkar*†‡, Igor Dodevski*†, Manca Kenig*§, Stefan Dudli*, Anja Mohr*, Emmanuel Hermans¶, and Andreas Plu¨ckthun*ʈ *Biochemisches Institut, Universita¨t Zu¨ rich, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland; and ¶Laboratoire de Pharmacologie Expe´rimentale, Universite´catholique de Louvain, Avenue Hippocrate 54.10, B-1200 Bruxelles, Belgium. Edited by William F. DeGrado, University of Pennsylvania, Philadelphia, PA, and approved July 18, 2008 (received for review April 1, 2008) We outline a powerful method for the directed evolution of sequence of a GPCR, keeping all other variables constant, to integral membrane proteins in the inner membrane of Escherichia yield more functionally expressed protein in a convenient het- coli. For a mammalian G protein-coupled receptor, we arrived at a erologous host, Escherichia coli. We used as a model system the sequence with an order-of-magnitude increase in functional ex- rat neurotensin receptor-1 (NTR1), which has been shown to pression that still retains the biochemical properties of wild type. give a detectable yield in E. coli (10, 11) but which still needs to This mutant also shows enhanced heterologous expression in be improved to allow more convenient preparation of milligram eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) quantities of receptor. and greater stability when solubilized and purified, indicating that Detailed characterization of the best variant from the selec- the biophysical properties of the protein had been under the tion reported here reveals that it exhibits an order-of-magnitude pressure of selection. These improvements arise from multiple increase in expression level in both E. coli and Pichia pastoris, small contributions, which would be difficult to assemble by elevated expression in mammalian cells, and enhanced stability rational design. In a second screen, we rapidly pinpointed a single in detergent-solubilized form, yet it largely retains the biochem- amino acid substitution in wild type that abolishes antagonist ical properties of WT NTR1, including binding affinity, binding binding while retaining agonist-binding affinity. These approaches selectivity, and G protein-mediated signaling. We have further may alleviate existing bottlenecks in structural studies of these extended this approach to isolate mutants of NTR1 with altered targets by providing sufficient quantities of stable variants in ligand selectivity. This methodology should thus be of general defined conformational states. utility in the directed evolution of stable variants of such proteins to high-level expression in multiple states of activity. integral membrane proteins ͉ protein engineering ͉ protein folding Results and Discussion protein-coupled receptors (GPCRs) comprise Ϸ1% of the Setup and Optimization of Screening Methodology. The general Ggenes in mammalian genomes and constitute Ϸ60% of all approach is given in Fig. 1. The expression vector containing the drug targets (1). Given the critical importance of this class of GPCR library of interest (e.g., from an error-prone PCR of the integral membrane proteins, there is great interest in having receptor gene) with two constant fusion partners (N-terminal detailed structures of these molecules. However, in the Protein maltose binding protein and C-terminal thioredoxin) is used to Data Bank (2), which contains Ϸ18,000 nonredundant protein express the corresponding proteins in functional form in the ␣ structures, the structures of only two GPCRs have been deposited, inner membrane of E. coli DH5 (see Methods). After expres- that of bovine rhodopsin (3), uniquely facilitated by its natural sion, cells are incubated at 4°C in an optimized buffer that abundance in the retina (4), and very recently that of ␤2 adrenergic renders the outer membrane permeable to small molecules to receptor (5), whose high resolution structure determination re- allow binding of fluorescent ligand to the receptors, and at the quired the replacement of a loop by a whole protein (6). same time maximizes cell viability (see Methods). Chen and Because most GPCRs are normally produced at very low colleagues (8) have previously described conditions in which levels, an overexpression system must be set up for each GPCR ligands as large as 10 kDa can enter the periplasmic space of E. of interest to obtain quantities sufficient for biophysical analyses. coli without compromising cell viability. Here, the buffer was Many attempts have been made to increase functional mem- specifically optimized to allow for saturable, specific binding of brane protein expression levels by testing various combinations fluorescently labeled agonist [BODIPY-NT(8–13)] to NTR1, of variables defining the expression system, such as host organ- while maximizing cell viability after fluorescence-activated cell ism, expression plasmid, fusion adducts, codon usage, and sorting (FACS) [see supporting information (SI) Figs. S1–S4]. expression induction conditions (7). However, even if all of these parameters could be globally optimized, they still may not yield Author contributions: C.A.S., I.D., E.H., and A.P. designed research; C.A.S., I.D., M.K., S.D., enough functional material because of inherent limitations in the and A.M. performed research; E.H. contributed new reagents/analytic tools; C.A.S., I.D., protein sequence itself. Furthermore, the limited stability of M.K., A.M., E.H., and A.P. analyzed data; and C.A.S., I.D., M.K., A.M., E.H., and A.P. wrote solubilized GPCRs is another bottleneck for their biophysical the paper. investigation, and this cannot be influenced by the expression The authors declare no conflict of interest. system. Thus, while it may be possible to solve a limited number This article is a PNAS Direct Submission. of new GPCR structures whose sequences are sufficiently ac- See Commentary on page 14747. commodating to support such a brute-force screening for ex- †C.A.S. and I.D. contributed equally to this work. pression and solubilization conditions, this method is unlikely to ‡Present address: Departments of Bioengineering and Chemical and Biomolecular Engi- have general applicability in routinely generating large amounts neering, University of Pennsylvania, 240 Skirkanich Hall, 210 South 33rd Street, Philadel- of stable, solubilized protein for any given GPCR. phia, PA 19104-6321. To directly address the importance of receptor sequence as an §Present address: Lek Pharmaceuticals, Kolodvorska 27, SI-1234 Menges, Slovenia. experimental variable in membrane protein expression and ʈTo whom correspondence should be addressed. E-mail: [email protected]. stability, we have developed a powerful approach, inspired by This article contains supporting information online at www.pnas.org/cgi/content/full/ periplasmic expression with cytometric screening (8) and an- 0803103105/DCSupplemental. chored periplasmic expression (9), in which we evolve the © 2008 by The National Academy of Sciences of the USA 14808–14813 ͉ PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803103105 Downloaded by guest on October 1, 2021 Table 2. KD of full-length neurotensin binding to receptors on whole cells SEE COMMENTARY NTR1 D03 E. coli 0.14 Ϯ 0.01 nM 0.11 Ϯ 0.01 nM HEK293T 3.7 Ϯ 0.5 nM 1.9 Ϯ 0.2 nM for the next selection. The flowchart for the selections on NTR1 is given in Fig. S5. Selection of Variants with Increased Expression Level. For increasing expression level, the initial randomized NTR1 library was sub- jected to four rounds of FACS. In each round, only the most fluorescent Ϸ0.1 to 1% of the cells were collected. Nonetheless, after these rounds, the evolved pool had a mean fluorescence intensity (MFI) no greater than that of the WT sequence. Error-prone PCR (epPCR) was used to overlay another set of random mutations on top of those that were enriched after the first four rounds of FACS, and this rerandomized library was again subjected to four rounds of sorting. In this second set of sorts, the MFI of the pool overtook that of WT NTR1. After a third randomization step followed by four more rounds of FACS, the evolved pool was split into two. One half was randomized by epPCR a fourth time and the other half was shuffled, using the staggered extension process (StEP) (12). After these selections, the MFI was approximately five times SCIENCES that of WT NTR1. From the enriched pool, 96 single clones were APPLIED BIOLOGICAL sequenced and analyzed for receptor expression level (see Fig. 1. General selection scheme for increasing expression level (steps 1, 2, Methods and Figs. S6 and S7). The clone with the best functional 3a, 4, back to 2) and altering ligand selectivity (steps 1, 2, 3b, 4, back to 2). receptor expression level per cell, D03, exhibited approximately a 10-fold increase in specific signal, as assayed by [3H]-NT binding and flow cytometry (Table 1 and Fig. S8). D03 has 14 Although the expression conditions for WT NTR1 typically nucleotide substitutions scattered throughout its helices and generate Ͻ1,000 functional receptors per cell (10, 11), optimi- loops (see Fig. S10 for a snake-like plot with the exact mutational zation of the binding buffer and FACS gating conditions resulted positions). Five of these mutations are silent, suggesting that in a specific signal in the gating window that was Ϸ900-fold above incorporation of nonsilent mutations was slow, approximately background (see Fig. S4). two amino acid substitutions per round of epPCR. This may be After incubation with saturating concentrations of BODIPY- due to the seven-transmembrane helical topology of the protein, which limits the number and type of mutations that are possible, NT(8–13), bacteria expressing the largest number of functional mandating a strategy with low mutational load. receptors correspondingly exhibit the greatest fluorescence, and these cells were collected directly in growth medium and then Evolved Receptor Retains Biochemical and Pharmacological Proper- expanded for a subsequent round.