Genetic immobilization of RNase Rny1p at the Saccharomyces cerevisiae cell surface

Teparić, Renata; Didak, Blanka; Ščulac, Elena; Mrša, Vladimir

doi:10.2323/jgam.59.75

Cited by 4 publications

(1 citation statement)

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“…Genetic immobilizations of S. cerevisiae RNase Rny1 and xylose reductase (XR) were achieved by fusions of the Ccw12 signal sequence followed by the catalytic part of the Rny1 or XR, respectively, and the GPI-anchoring signal of the Ccw12 [60,61].…”

Section: Recombinant Protein Fusion To Other Gpi-anchored Proteinsmentioning

confidence: 99%

Surface Display—An Alternative to Classic Enzyme Immobilization

et al. 2019

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Enzyme immobilization to solid matrices often presents a challenge due to protein conformation sensitivity, desired enzyme purity, and requirements for the particular carrier properties and immobilization technique. Surface display of enzymes at the cell walls of microorganisms presents an alternative that has been the focus of many research groups worldwide in different fields, such as biotechnology, energetics, pharmacology, medicine, and food technology. The range of systems by which a heterologous protein can be displayed at the cell surface allows the appropriate one to be found for almost every case. However, the efficiency of display systems is still quite low. The most frequently used yeast for the surface display of proteins is Saccharomyces cerevisiae. However, apart from its many advantages, Saccharomyces cerevisiae has some disadvantages, such as low robustness in industrial applications, hyperglycosylation of some heterologous proteins, and relatively low efficiency of surface display. Thus, in the recent years the display systems for alternative yeast hosts with better performances including Pichia pastoris, Hansenula polymorpha, Blastobotrys adeninivorans, Yarrowia lipolytica, Kluyveromyces marxianus, and others have been developed. Different strategies of surface display aimed to increase the amount of displayed protein, including new anchoring systems and new yeast hosts are reviewed in this paper.

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Section: Recombinant Protein Fusion To Other Gpi-anchored Proteinsmentioning

confidence: 99%

Surface Display—An Alternative to Classic Enzyme Immobilization

et al. 2019

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Measuring the capacity of yeast for surface display of cell wall‐anchored protein isoforms by using β‐lactamase as a reporter enzyme

Martinić Cezar,

Paić,

Prekpalaj

et al. 2024

FEBS Open Bio

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Yeast surface display is a promising biotechnological tool that uses genetically modified yeast cell wall proteins as anchors for enzymes of interest, thereby transforming yeast cell wall into a living catalytic material. Here, we present a comprehensive protocol for quantifying surface‐displayed β‐lactamase on the cell wall of model yeast Saccharomyces cerevisiae. We use β‐lactamase as a reporter enzyme, which we tagged to be anchored to the cell wall closer to its N or C terminus, through the portion of the Pir2 or Ccw12 cell wall proteins, respectively. The catalytic activity of surface‐displayed β‐lactamase is assessed by its ability to hydrolyze nitrocefin, which produces a colorimetric change that is quantitatively measured by spectrophotometric analysis at 482 nm. This system enables precise quantification of the potential of S. cerevisiae strains for surface display, continuous real‐time monitoring of enzyme activity, and facilitates the study of enzyme kinetics and interactions with inhibitors within the cell's native environment. In addition, the system provides a platform for high‐throughput screening of potential β‐lactamase inhibitors and can be adapted for the visualization of other enzymes, making it a versatile tool for drug discovery and bioprocess development.

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