scholarly journals Affinity maturation: highlights in the application of in vitro strategies for the directed evolution of antibodies

2021 ◽  
Author(s):  
Denice T.Y. Chan ◽  
Maria A.T. Groves
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Sequence Space ◽  
Therapeutic Potential ◽  
Dna Recombination ◽  
Affinity Maturation ◽  
Therapeutic Antibodies ◽  
Effective Strategies ◽  
Key Features

Affinity maturation is a key technique in protein engineering which is used to improve affinity and binding interactions in vitro, a process often required to fulfil the therapeutic potential of antibodies. There are many available display technologies and maturation methods developed over the years, which have been instrumental in the production of therapeutic antibodies. However, due to the inherent limitations in display capacity of these technologies, accommodation of expansive and complex library builds is still a challenge. In this article, we discuss our recent efforts in the affinity maturation of a difficult antibody lineage using an unbiased approach, which sought to explore a larger sequence space through the application of DNA recombination and shuffling techniques across the entire antibody region and selections using ribosome display. We also highlight the key features of several display technologies and diversification methods, and discuss the strategies devised by different groups in response to different challenges. Particular attention is drawn to examples which are aimed at the expansion of sequence, structural or experimental diversity through different means and approaches. Here, we provide our perspectives on these methodologies and the considerations involved in the design of effective strategies for the directed evolution of antibodies.

In vitro affinity maturation of HuCAL antibodies: complementarity determining region exchange and RapMAT technology

Immunotherapy ◽  
2009 ◽  
Vol 1 (4) ◽  
pp. 571-583
Author(s):  
Josef Prassler ◽  
Stefan Steidl ◽  
Stefanie Urlinger
Keyword(s):  
Monoclonal Antibodies ◽  
Affinity Maturation ◽  
Optimization Techniques ◽  
Human Diseases ◽  
Therapeutic Antibodies ◽  
Naturally Occurring ◽  
Human Immunoglobulins ◽  
Antibody Libraries ◽  
Complementarity Determining Region

Monoclonal antibodies gain ever-increasing importance in the treatment of human diseases across a broad range of indications. Diverse technologies currently exist, which are used to generate recombinant therapeutic antibodies that are basically indistinguishable from naturally occurring human immunoglobulins. We describe how human combinatorial antibody libraries are used together with unique optimization techniques to produce such therapeutically relevant proteins, for instance in the areas of oncology and inflammation.

A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution

Lab on a Chip ◽  
2012 ◽  
Vol 12 (5) ◽  
pp. 882 ◽  
Author(s):  
Ali Fallah-Araghi ◽  
Jean-Christophe Baret ◽  
Michael Ryckelynck ◽  
Andrew D. Griffiths
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Screening System

scholarly journals Protein engineering of fungal xylanase

2007 ◽  
Author(s):  
◽  
Dawn Elizabeth Stephens
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Chlorine Dioxide ◽  
Dna Recombination ◽  
Thermomyces Lanuginosus ◽  
Dna Shuffling ◽  
Genetic Level ◽  
The Creation ◽  
Pre Treatment ◽  
Paper Pulps

Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.

scholarly journals Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

2015 ◽  
Vol 81 (18) ◽  
pp. 6451-6462 ◽  
Author(s):  
Javier Viña-Gonzalez ◽  
David Gonzalez-Perez ◽  
Patricia Ferreira ◽  
Angel T. Martinez ◽  
Miguel Alcalde
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Signal Sequence ◽  
Dna Recombination ◽  
Random Mutagenesis ◽  
Killer Toxin ◽  
Alcohol Oxidase ◽  
Screening Assay ◽  
Content Type

ABSTRACTAryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO fromPleurotus eryngiiwas replaced by those of the mating α-factor and the K1killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion inSaccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: preproα-AAO > preαproK-AAO > preKproα-AAO > preproK-AAO. The chimeric preαproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[α1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preαproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except forP. eryngiiandP. pulmonariusAAO. Thein vitroinvolution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.

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