2, mAbs RG-1, K18L2(20C38), and K4L2(20C38) efficiently and specifically bound the recombinant X-L2 particles in all genetic backgrounds. wild-type amino acid, PSI-6206 13CD3 position, substituted amino acid. Thus, A83D, an alanine to a aspartic acid at residue 83. fThese phages contained mutations within the HA insert as well as in G protein. The changes in the insert were Y5F, Y10H, D9G. Numbering of the insert is the same as given in Table 2. Table 2 Amino acid insertions placed at position Thr21 of G protein. CS protein160GSNANPNANPNANPSGYesCScCS protein240GSNANPNANPNANPNANPNANPSGNoCSdCS protein280GSNANPNANPNANPNANPNANPNANPSGNoCSeCS protein28?1GSNANPNVDPNANPNANPNANPNANPSGYes Open in a separate window aIndicates whether the insertion was tolerated within the phage genome or yielded a complementing clone. The PTSCD insertion was too large to be moved into the genome, see text for details. Although it is possible to conduct complementation assays with expression plasmids made up of the cloned wild-type X174G gene, induction is usually somewhat toxic to host cells. Modified gene G induction conferred two distinct phenotypes, neither of which resembled the wild-type control (Table 1). The induction of the cloned XG21-L2 gene killed host cells, whereas XG47-L2, XG151-L2 and XG153-L2 gene induction resulted in no observable toxicity. Unlike the wild-type G gene, these recombinant genes failed to complement the X174 am (G)Q33 mutant. The elevated or decreased toxicity levels proved a very reliable readout for G protein functionality (see below). To determine if altered genes could be placed directly into viable phage genomes, recombination rescue experiments PSI-6206 13CD3 were performed. X174 am((Gambhira et al., 2007; Murtif et al., 1985; Reddy et al., 2000; Wang et al., 1996; Young et al., 2012; Zavala et al., 1985; Zebedee and Lamb, 1988). These inserts encompass a wide variety of sizes, charges, and side chain chemistries (Table 2). Cloned genes were assayed for elevated host cell toxicity upon induction and the ability to complement X174 defect affects protein folding and/or is usually manifested during early assembly. The presence of smaller assembly intermediates was examined by SDS-PAGE of gradient fractions within the 6S-12S range, which contain the early assembly intermediates (Fig. 1D). Unlike PSI-6206 13CD3 the wild-type control, 12S* particles were not detected in X-HA infected cell extracts (Fig. 1C). This particle contains the F, G, B and H proteins in a respective 5:5:5:1 ratio. It is formed by the addition of one G protein pentamer (6S particle) to the 9S* intermediate. 6S gradient fractions, containing soluble G protein pentamers were compared side by side on PSI-6206 13CD3 an SDS-PAGE gel (Fig. 1E). The intensity of the G protein band was normalized to a host protein band, which is present in both cell lysates and 6S fractions and indicated in Fig. 1E. In whole cell lysates, this protein G:host protein ratio measures total cellular G protein, regardless of solubility. Protein G was abundant in both the wild-type and XHA whole cell lysates with ratios of 2.7 and 1.6, respectively. The G protein levels in the 6S fraction roughly estimates the amount of folded soluble protein. If mutant 6S particles form but are not incorporated into 12S* particles, they would accumulate in this fraction. Within the 6S fraction, the wild-type protein G: host protein ration is 1.5. Rabbit Polyclonal to C-RAF (phospho-Ser301) By contrast, the X-HA protein G:host protein ratio is only 0.2. Thus, X-HA 6S particles appear to be vastly underrepresented when compared to the wild-type control. These data indicate that the HA recombinant G protein folds poorly at 42 C and is unable to assemble into 6S* pentamers. Second-site suppressors within G correct for high-temperature growth defects Suppressors of the ts phenotype were selected by plating XHA, X-M2b, X-M2c, and X-CSa and X-L2 at 42 C, as described in Materials and Methods. Seven independent phage preparations were used to isolate suppressors of the X-L2 ts growth defect. The A83V mutation was isolated thrice independently, whereas the A83D mutation was isolated twice independently. Thus, the selection likely approached saturation (Luria and Delbruck, 1943). Other suppressors were located at Thr105 and Ala106 (Table 1). These substitutions affect two different insertion loops within the G protein (Fig. 1B). In less exhaustive selections, substitutions for A83 were isolated in the X-M2b, X-M2c, and X-CSa backgrounds. The suppressor of the X-HA ts phenotype were located directly within the HA insertion (Tables ?(Tables11 and ?and2).2). However, one of these substitutions HA-D9G was always isolated in conjunction with either the A83V or A106D mutations. These results indicate that most folding defects can be corrected by global suppressors located too far away from the insertion to directly alter antigen display..
