Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL

Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

doi:10.1007/978-1-4939-6472-7_28

Cited by 2 publications

(2 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(described in detail below) [22], who tailored a novel sialic acid-binding lectin from an R-type galactose-binding protein using random mutagenesis and a ribosome-display method under the concept of 'natural evolution-mimicry' [23]. Since their work, a significant number of reports of protein engineering base lectin engineering have come out of different laboratories [24][25][26][27][28][29][30][31][32][33][34][35][36]. Aptamer and chemistry-based lectin engineering are also promising and challenging.…”

Section: Current State Of the Art Of Lectin Engineeringmentioning

confidence: 99%

Lectin engineering: the possible and the actual

Hirabayashi

Arai

2019

Interface Focus.

View full text Add to dashboard Cite

Lectins are a widespread group of sugar-binding proteins occurring in all types of organisms including animals, plants, bacteria, fungi and even viruses. According to a recent report, there are more than 50 lectin scaffolds (∼Pfam), for which three-dimensional structures are known and sugar-binding functions have been confirmed in the literature, which far exceeds our view in the twentieth century (Fujimoto et al. 2014 Methods Mol. Biol. 1200 , 579–606 ( doi:10.1007/978-1-4939-1292-6_46 )). This fact suggests that new lectins will be discovered either by a conventional screening approach or just by chance. It is also expected that new lectin domains including those found in enzymes as carbohydrate-binding modules will be generated in the future through evolution, although this has never been attempted on an experimental level. Based on the current state of the art, various methods of lectin engineering are available, by which lectin specificity and/or stability of a known lectin scaffold can be improved. However, the above observation implies that any protein scaffold, including those that have never been described as lectins, may be modified to acquire a sugar-binding function. In this review, possible approaches to confer sugar-binding properties on synthetic proteins and peptides are described.

show abstract

Section: Current State Of the Art Of Lectin Engineeringmentioning

confidence: 99%

Lectin engineering: the possible and the actual

Hirabayashi

Arai

2019

Interface Focus.

View full text Add to dashboard Cite

show abstract

“…The mutagenesis approach is used for the elucidation of sugar binding mechanisms of lectins [8] as well as for the modification of lectin properties [9, 10]. The functional characterization of lectins and their mutant variants is performed by methods such as isothermal titration calorimetry, surface plasmon resonance, enzyme-linked lectin assay, microscale thermophoresis, or glycan arrays [11–13].…”

Section: Introductionmentioning

confidence: 99%

Microscopy examination of red blood and yeast cell agglutination induced by bacterial lectins

2019

Self Cite

View full text Add to dashboard Cite

Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope.

show abstract

Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL

Cited by 2 publications

References 39 publications

Lectin engineering: the possible and the actual

Lectin engineering: the possible and the actual

Microscopy examination of red blood and yeast cell agglutination induced by bacterial lectins

Contact Info

Product

Resources

About