RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

BACKGROUND: In 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. RESULTS: We 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 V(NAR )shark antibody fragment (12Y-2) against malarial apical membrane antigen-1 (AMA-1) via ribosome display. The binding constant (K(D)) 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. CONCLUSION: The 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

A Novel Nanobody Specific for Respiratory Surfactant Protein A has Potential for Lung Targeting

Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies\u27 potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery

Biopharming the SimpliRED™ HIV diagnostic reagent in barley, potato and tobacco

This is the first report of an antibody-fusion protein expressed in transgenic plants for direct use in a medical diagnostic assay. By the use of gene constructs with appropriate promoters, high level expression of an anti-glycophorin single-chain antibody fused to an epitope of the HIV virus was obtained in the leaves and stems of tobacco, tubers of potato and seed of barley. This fusion protein replaces the SimpliRED™ diagnostic reagent, used for detecting the presence of HIV-1 antibodies in human blood. The reagent is expensive and laborious to produce by conventional means since chemical modifications to a monoclonal antibody are required. The plant-produced fusion protein was fully functional (by ELISA) in crude extracts and, for tobacco at least, could be used without further purification in the HIV agglutination assay. All three crop species produced sufficient reagent levels to be superior bioreactors to bacteria or mice, however barley grain was the most attractive bioreactor as it expressed the highest level (150 μg of reagent g-1), is inexpensive to produce and harvest, poses a minuscule gene flow problem in the field, and the activity of the reagent is largely undiminished in stored grain. This work suggests that barley seed will be an ideal factory for the production of antibodies, diagnostic immunoreagents, vaccines and other pharmaceutical proteins

Quantitative guidelines for the prediction of ultrasound contrast agent destruction during injection

Experiments and theory were undertaken on the destruction of ultrasound contrast-agent microbubbles on needle injection, with the aim of predicting agent loss during in-vivo studies. Agents were expelled through a variety of syringe and needle combinations, subjecting the microbubbles to a range of pressure-drops. Imaging of the bubbles identified cases where bubbles were destroyed and the extent of destruction. Fluid-dynamical calculations determined the pressure-drop for each syringe and needle combination. It was found that agent destruction occurred at a critical pressure-drop that depended only on the type of microbubble. Protein-shelled microbubbles (sonicated bovine serum albumin) were virtually all destroyed above their critical pressure-drop of 109 +/- 7 kPa. Two types of lipid-shelled microbubbles were found to have a pressure-drop threshold above which more than 50% of the microbubbles were destroyed. The commercial lipid-shelled agent Definity was found to have a critical pressure-drop for destruction of 230 +/- 10 kPa, and for a previously-published lipid-shelled agent, it was 150 +/- 40 kPa. It is recommended that attention to the predictions of a simple formula could preclude unnecessary destruction of microbubble contrast agents during in vivo injections. This approach may also preclude undesirable release of drug or gene payloads in targeted microbubble therapies. Example values of appropriate injection rates for various agents and conditions are given

RNA mutagenesis yields highly diverse mRNA libraries for <it>in vitro </it>protein evolution

Abstract Background In 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. Results We 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. Conclusion The 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.</p

Sugar analog synthesis by <i>in vitro</i> biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis - Fig 1

<p><b>Aldolase enzymes are involved in both gluconeogenesis and glycolysis (a) and are classified into two classes by two distinct mechanisms of action for nucleophilic activation</b>. Class I aldolases exhibit a strictly conserved lysine residue which forms a covalent Schiff base with the donor molecule to generate an enamine nucleophile (b) whilst class II aldolases use a divalent metal cation cofactor to promote enolization of the donor molecule <i>via</i> bidentate Lewis acid complexation (c).</p

DHAP production <i>via</i> two step enzymatic cascade.

<p>DHAP production <i>via</i> two step enzymatic cascade.</p

Details of the optimized cascade for the production of DHAP from glycerol.

<p>Phosphorylation of glycerol by ATP mediated by GlpK_<i>Tk</i> (EC 2.7.1.30) and Mg²⁺ <i>via</i> a phosphotransfer mechanism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184183#pone.0184183.ref033" target="_blank">33</a>] was accompanied by regeneration of ATP from ADP by AceK_<i>Ms</i> (EC 2.7.2.1), which catalyzes reversibly the phosphorylation of acetate in the presence of a divalent cation and ATP with the formation of acetylphosphate and ADP[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184183#pone.0184183.ref034" target="_blank">34</a>]. Cytosolic glycerophosphate oxidase GlpO_<i>Mg</i>(EC 1.1.3.21) likely converts glycerol-3-phosphate to DHAP by a similar mechanism to the related GlpO from <i>Mycoplasma pneumoniae</i> (4X9M) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184183#pone.0184183.ref035" target="_blank">35</a>]), Similarly to other flavoprotein oxidases, glycerophosphate oxidase GlpO enzymes follow a hydride transfer mechanism to stabilize a positive charge on the flavin N(5)-sulfite adduct (C). The hydrogen peroxide generated from the oxidation of enzymatic FADH₂ was converted to water by the addition of catalase from <i>Micrococcus lysodeikticus</i>.</p

Production of rare chiral sugars by combining optimized multi-enzyme cascades for DHAP production with a DHAP-dependant fructose-1,6-biphosphate aldolase.

<p>Glycerol (10mM substrate) was converted to glycerol-3-phosphate by a glycerol kinase enzyme GlpK_<i>Tk</i> (28.6 pmoles) with concomitant regeneration of ATP by an acetate kinase enzyme AceK_<i>Ms</i> (40.2 pmoles). The glycerol-3-phopshate was then oxidized to DHAP by a novel <i>L</i>-glycerol-3-phosphate oxidase enzyme GlpO_<i>Mg</i> (154.2 pmoles), with mitigation of excess hydrogen peroxide by catalase (3U/mL) and an aldolase enzyme FruA_<i>Sc</i> (3.1 nmoles) converted this and acceptor aldehydes (provided at 10mM) into chiral sugars <i>D</i>-fructose-1,6-biphosphate (3<i>S</i>, 4<i>R</i>) and 3,4-dihydroxyhexulose phosphate (3<i>S</i>, 4<i>R</i>) as depicted.</p