Abstract

BackgroundIn protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qβ replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal.ResultsWe show that Qβ replicase generates all possible base substitutions with an equivalent preference for mutating A/T or G/C bases and with no significant bias for transitions over transversions. To demonstrate the high diversity that can be sampled from a Qβ replicase-generated mRNA library, the approach was used to evolve the binding affinity of a single domain VNAR shark antibody fragment (12Y-2) against malarial apical membrane antigen-1 (AMA-1) via ribosome display. The binding constant (KD) of 12Y-2 was increased by 22-fold following two consecutive but discrete rounds of mutagenesis and selection. The mutagenesis method was also used to alter the substrate specificity of β-lactamase which does not significantly hydrolyse the antibiotic cefotaxime. Two cycles of RNA mutagenesis and selection on increasing concentrations of cefotaxime resulted in mutants with a minimum 10,000-fold increase in resistance, an outcome achieved faster and with fewer overall mutations than in comparable studies using other mutagenesis strategies.ConclusionThe RNA based approach outlined here is rapid and simple to perform and generates large, highly diverse populations of proteins, each differing by only one or two amino acids from the parent protein. The practical implications of our results are that suitable improved protein candidates can be recovered from in vitro protein evolution approaches using significantly fewer rounds of mutagenesis and selection, and with little or no collateral damage to the protein or its mRNA.

Highlights

  • IntroductionIn vitro molecular optimization or protein maturation can be used to modify protein properties

  • In protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties

  • As Qβ phage has a double-stranded RNA genome, Qβ replicase has a strong bias for replicating its own genome with both RNA (-) and (+) strands serving as templates [14,15]

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Summary

Introduction

In vitro molecular optimization or protein maturation can be used to modify protein properties. There is a growing demand by the pharmaceutical and medical industries for protein molecules, including antibodies, of diagnostic and therapeutic efficacy, as well as a perpetual need in the production and manufacturing industries for improved biocatalysts These demands have directed the innovation of a number of sophisticated and complex methods for the in vitro evolution and optimization of proteins [1]. One fundamental approach to this process is the introduction of random mutations into a known nucleotide sequence to produce a library of variants These variants are subsequently translated to produce modified proteins that are screened for chosen properties. The potential of this approach has been limited by deficiencies in the methods currently available for random mutagenesis and library generation [2]. Directed protein evolution using powerful selection strategies such as ribosome display (described below) are more likely to identify improved variants when a library is maximally diverse which would be the case when all variants in a library are probable [10]

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