Paul.xu: Created page with "A 12Y-2 variant library was created by mutating the gene
sequence at the RNA level using Qbeta replicase (G.K., submitted
for publication). Briefly, mRNA template..."

2019-07-15T18:40:00Z

Created page with "A 12Y-2 variant library was created by mutating the gene sequence at the RNA level using Qbeta replicase (G.K., submitted for publication). Briefly, mRNA template..."

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

Affinity maturation using qbeta replicase - Revision history

Paul.xu: Created page with "A 12Y-2 variant library was created by mutating the genesequence at the RNA level using Qbeta replicase (G.K., submittedfor publication). Briefly, mRNA template..."

Paul.xu: Created page with "A 12Y-2 variant library was created by mutating the gene
sequence at the RNA level using Qbeta replicase (G.K., submitted
for publication). Briefly, mRNA template..."