Palmitoylation

The β₁AR construct T34-424/His642 (link) was the starting point for the generation of the β₁AR36-m23 construct that crystallized. The C-terminus was further truncated after Leu367, and 6 histidines were added. Two segments, comprising residues 244-271 and 277-278 of CL3, were also deleted. The construct included the following 8 point mutations: C116^3.27L increased expression; C358A at the C-terminus of H8 removed palmitoylation and helped crystallisation; R68^1.59S, M90^2.53V, Y227^5.58A, A282^6.27L, F327^7.37A and F338^7.48M thermostabilised the receptor in the antagonist conformation15 (link). Baculovirus expression and receptor purification42 (link) were performed in decylmaltoside, with the detergent exchanged to octylthioglucoside on the alprenolol sepharose column. Crystals were obtained by vapour diffusion at 18°C with hanging drops after addition of an equal volume of reservoir solution, 0.1M N-(2-acetamido)iminodiacetic acid:NaOH pH 6.9-7.3 and 29-32% PEG600 to receptor concentrated to 6.0 mg/ml.

The rAAV-Cap-in-cis-lox genome plasmid contains three main elements flanked by AAV2 ITRs: (i) an mCherry expression cassette, which is comprised of a 398bp fragment of the human UBC gene upstream of the mCherry cDNA followed by a synthetic polyadenylation sequence^{40 (link)}; (ii) the AAV9 capsid gene and regulatory sequences, which are comprised of the AAV5 p41 promoter sequence (1680-1974 of GenBank AF085716.1)^{41 (link),42 (link)} and splicing sequences taken from the AAV2 rep gene; and (iii) a Cre-dependent switch, which is comprised of the SV40 polyadenylation sequence (pA) flanked by inverted lox71 and lox66 sites^{43 (link)} (Fig. 1b). The rAAV-Cap-in-cis-lox genome plasmid was further modified to introduce two unique restriction sites, XbaI and AgeI, within the capsid sequence. These sites flank the region (AA450-592) that is replaced by the randomized library fragment. The introduction of the XbaI site introduces a K449R mutation, which does not have an overt effect on vector production or transduction. The mutations required to insert the AgeI site are silent. For the rAAV-ΔCap-in-cis acceptor plasmid used for the capsid library cloning, the coding region between the XbaI and AgeI sites was removed to prevent virus production from the acceptor plasmid lacking the library fragment.
As a template for the library fragment, we PCR amplified the region spanning the XbaI and AgeI sites of the modified AAV9. This sequence was modified to remove a unique EarI restriction site and insert a unique KpnI site (both silent mutations) to create the xE fragment. The modified xE fragment was TA cloned into pCRII (Life Technologies) to generate pCRII-9Cap-xE. Eliminating the EarI site provided a second method that could be used, if necessary, to selectively digest contaminating (AAV9) capsid sequences recovered by PCR, but not digest the library-derived sequences. We did not find it necessary to use this digestion step. Using the rAAV-ΔCap-in-cis acceptor for cloning the libraries and taking standard PCR precautions (e.g., UV treating reagents and pipettors) was sufficient to prevent contamination.
The AAV2/9 REP-AAP helper plasmid was constructed by introducing five stop codons into the coding sequence of the VP reading frame of the AAV9 gene at VP1 AAs: 6, 10, 142, 148 and 216. The stop codon at AA216 was designed not to disrupt the coding sequence of the AAP protein, which is encoded within an alternative reading frame.
Several rAAV genomes were used in this study. Each is constructed within a single stranded (ss) rAAV genome with a reporter driven by the ubiquitous CMV-β-Actin-intron-β-Globin hybrid promoter (CAG). For simplicity, the vector descriptions have been abbreviated in the text. ssAAV-CAG-GFP refers to ssAAV-CAG-eGFP-2A-Luc-WPRE-SV40 polyA. ssAAV-CAG-NLS-GFP refers to ssAAV-CAG-NLS-GFP-WPRE-SV40 polyA, which was constructed by inserting the nuclear localization sequence PKKKRKV at both the N- and C-termini of GFP. ssAAV-CAG-mNeGreen-f refers to ssAAV-CAG-mNeonGreen-f-WPRE with a human growth hormone polyA signal. The mNeonGreen^{44 (link)} was modified with the membrane targeting (farnesylation and palmitoylation signals) sequence from c-Ha-Ras^{45 (link)}.