Pardo, Isabel Camarero, Susana
Published in
Methods in molecular biology (Clifton, N.J.)
In this chapter we describe several high-throughput screening assays for the evaluation of mutant libraries for the directed evolution of fungal laccases in the yeast Saccharomyces cerevisiae. The assays are based on the direct oxidation of three syringyl-type phenols derived from lignin (sinapic acid, acetosyringone, and syringaldehyde), an artifi...
Wijma, Hein J Fürst, Maximilian J L J Janssen, Dick B
Published in
Methods in molecular biology (Clifton, N.J.)
The ability to stabilize enzymes and other proteins has wide-ranging applications. Most protocols for enhancing enzyme stability require multiple rounds of high-throughput screening of mutant libraries and provide only modest improvements of stability. Here, we describe a computational library design protocol that can increase enzyme stability by 2...
Fulton, Alexander Hayes, Marc R Schwaneberg, Ulrich Pietruszka, Jörg Jaeger, Karl-Erich
Published in
Methods in molecular biology (Clifton, N.J.)
Screening is defined as the identification of hits within a large library of variants of an enzyme or protein with a predefined property. In theory, each variant present in the respective library needs to be assayed; however, to save time and consumables, many screening regimes involve a primary round to identify clones producing active enzymes. Su...
Arjumand, Wani Asiaf, Asia Ahmad, Shiekh Tanveer
Published in
Methods in molecular biology (Clifton, N.J.)
DNA repair machinery preserves genomic integrity, which is frequently challenged through endogenous and exogenous toxic insults, and any sort of repair machinery malfunctioning ultimately manifests in the form of several types of terrible human diseases such as cancers (Hoeijmakers, Nature 411(6835): 366-374, 2001). Noncoding RNAs (ncRNAs) are cruc...
Tutar, Lütfi Özgür, Aykut Tutar, Yusuf
Published in
Methods in molecular biology (Clifton, N.J.)
Our understanding of cancer pathways has been changed by the determination of noncoding transcripts in the human genome in recent years. miRNAs and pseudogenes are key players of the noncoding transcripts from the genome, and alteration of their expression levels provides clues for significant biomarkers in pathogenesis of diseases. Especially, miR...
Bitton, Almog Nahary, Limor Benhar, Itai
Published in
Methods in molecular biology (Clifton, N.J.)
Antibody libraries came into existence 25 years ago when the accumulating sequence data of immunoglobulin genes and the advent of the PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody li...
Shin, Daniel Nagarajan, Rajesh
Published in
Methods in molecular biology (Clifton, N.J.)
Bacteria use chemical molecules called autoinducers as votes to poll their numerical strength in a colony. This polling mechanism, commonly referred to as quorum sensing, enables bacteria to build a social network and provide a collective response for fighting off common threats. In Gram-negative bacteria, AHL synthases synthesize acyl-homoserine l...
Cao, Wei Cheng, Wenfang Wu, Wei
Published in
Methods in molecular biology (Clifton, N.J.)
Endogenously produced microRNAs (miRNAs) are predicted to regulate the translation of over two-thirds all human gene transcripts. Certain microRNAs regulate expression of genes that are critically involved in both innate and adaptive immune responses. miRNAs have been demonstrated to function as crucial regulators of immune response under both phys...
Avril, Arnaud Miethe, Sebastian Hust, Michael Pelat, Thibaut
Published in
Methods in molecular biology (Clifton, N.J.)
Rapidly after the clinical success of the first murine therapeutic antibody licensed in 1985 (muromomab-CD3), the first limits of the therapeutic use of antibodies deriving from hybridoma technology appeared. Indeed, the nonhuman nature of these therapeutic antibodies makes them immunogenic when administrated to patients, which develop anti-drug an...
Santos-Aberturas, Javier Dörr, Mark Bornscheuer, Uwe T
Published in
Methods in molecular biology (Clifton, N.J.)
The different expression level and solubility showed by each protein variant represents an important challenge during screening campaigns: Usually, the total activity measurement constitutes the only criterion for identifying improved variants. This hampers the chances of finding interesting mutants, especially if the aim is to improve activity: On...
