Editing Affinity maturation using qbeta replicase

A 12Y-2 variant library was created by mutating the gene<br/>sequence at the RNA level using Qbeta&nbsp;replicase (G.K., submitted<br/>for publication). Briefly, mRNA template suitable for&nbsp;replicase was transcribed from DNA template with T7 polymerase,<br/>DNase-treated to remove the DNA template (RQ1<br/>DNase, Promega) and purified (RNeasy, Qiagen). One hundred<br/>nanograms of the mRNA template was pre-heated for 2 min at<br/>95 ◦C in a thermocycler and permitted to slowly cool to room<br/>temperature. The RNA was mixed with 40mM Tris–HCl (pH<br/>7.9), 21mM MgCl2, 2mM spermidine, 10mM dithiothreitol,<br/>1mMeach of rCTP, rUTP, rGTP, and rATP, 2U RNase inhibitor<br/>(Promega) and 200nM Qbeta&nbsp;replicase (prepared in house following<br/>the method of Moody et al. [12]) and incubated for a<br/>minimum of 120 min at 37 ◦C. The Qbeta&nbsp;replicase amplified RNA<br/>was purified (RNeasy, Qiagen) to remove excess MgCl2 prior to<br/>ribosome display.<br/>2.3. Ribosome display<br/>Ribosome display was performed essentially as outlined<br/>[13,14]. Two micrograms of heat denatured Qbeta&nbsp;replicase<br/>mutated RNA was directly added to a 50uL rabbit reticulocyte<br/>based translation reaction (Promega) following the manufacturer’s<br/>recommendations. Translation was allowed to proceed<br/>for 25 min at 30 ◦C before diluting the translation mix with the<br/>addition of biotin-free skim milk (4%, w/v), PBS, and 5mM<br/>MgCl2. One nanomolar biotinylated AMA1 was added directly<br/>to the diluted translation mix which was subsequently rocked at<br/>4 ◦C for 5 h prior to the addition of 2000nM AMA1. The mix<br/>was rocked at 4 ◦C for a further 2 h (which constituted the competition<br/>phase). Ribosome complexes that remained attached to<br/>the biotinylated AMA1 were recovered with streptavidin coated<br/>magnetic beads (Dynal). Beads were subsequently washed three<br/>times with PBS containing 5mMMgCl2 and 0.1% Tween 20 and<br/>two times with PBS containing 5mM MgCl2 before being resuspended<br/>in 40 uL of pre-heated dH2O (55 ◦C) to disrupt the<br/>ribosome complexes. The beads were removed and the supernatant<br/>was used directly in RT-PCR to recover RNA using gene<br/>specific primers, the RT-PCR product cloned into expression<br/>vector pGC, and 500 individual clones analyzed for binding<br/>[15]. A second round of mutagenesis and ribosome display<br/>was performed on the best variant/s identified in round one,<br/>but incorporated increased selection pressure by increasing the<br/>competition phase to 16 h prior to RNA recovery. Following the<br/>second round of mutagenesis and panning, 500 individual clones<br/>were analyzed.
[[Category:Antibody]]