Affinity maturation using qbeta replicase

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.