Yeast Surface Display: New Opportunities for a Time-Tested Protein Engineering System

Raeeszadeh‐Sarmazdeh, Maryam; Boder, Eric T.

doi:10.1007/978-1-0716-2285-8_1

Cited by 11 publications

(7 citation statements)

References 136 publications

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“…The inhibition for minimal TIMP library was performed using the method for tight binding inhibition, as described previously (Radisky et al, 2017; Raeeszadeh‐Sarmazdeh & Boder, 2022; Raeeszadeh‐Sarmazdeh, Patel, et al, 2019). Briefly, MMP inhibition activities were determined via measuring the rate of MMP cleavage of fluorogenic MMP substrate, Mca‐Pro‐Leu‐Gly‐Leu‐Dap(Dnp)‐Ala‐Arg‐NH2 (AnaSpec) with a range of the inhibitor variants' concentrations.…”

Section: Methodsmentioning

confidence: 99%

“…To further unravel the key motifs of different human TIMP in binding and inhibition of specific MMPs, we generated a library of minimal TIMPs based on four full‐length human TIMPs using DNA shuffling on yeast surface. Yeast surface display has been proven to be a strong platform used for screening a library of mutants for various protein and peptide (Boder et al, 2012; Boder & Wittrup, 1997; Raeeszadeh‐Sarmazdeh & Boder, 2022; Raeeszadeh‐Sarmazdeh, Patel, et al, 2019) and has been previously used for engineering TIMPs (Arkadash et al, 2017). We screened the library of minimal TIMP variants toward binding to MMP‐3 and MMP‐9, a stromelysin and gelatinase, respectively, two soluble MMPs with distinct sequences, structure, and functions, using FACS.…”

Section: Introductionmentioning

confidence: 99%

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Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display

Hosseini,

Kumar,

Hedin

et al. 2023

Protein Science

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Overexpression of specific matrix metalloproteinases (MMPs) has a key role in development of several diseases, such as cancer, neurological disorders, and cardiovascular diseases due to their critical role in degradation and remodeling of the extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs), a family of four in human, are endogenous inhibitors of MMPs. TIMPs have a high level of sequence and structure homology, with a broad range of binding and inhibition to the family of MMPs. It is important to identify the key motifs of TIMPs responsible in inhibition of MMPs for developing efficient therapeutics targeting specific MMPs. We used DNA shuffling between the human TIMP family to generate a minimal TIMP hybrid library to identify the dominant minimal MMP inhibitory regions in yeast. The minimal TIMP variants screened toward MMP‐3 and MMP‐9 using fluorescent‐activated cell sorting (FACS). Interestingly, several minimal TIMP variants selected after screening toward MMP‐3cd or MMP‐9cd, with lengths as short as 20 amino acids, maintained or improved binding to MMP‐3 and MMP‐9. The TIMP‐MMP binding dissociation constant (KD), in the nM range, and MMP inhibition constants (Ki), in the pM range, of these minimal TIMP variants were similar to the N‐terminal domain of TIMP‐1 on the yeast surface and in solution indicating the potency of these minimal variants as MMP inhibitors. We further used molecular modeling simulation, and molecular protein docking of the minimal TIMP variants in complex with MMP‐3cd to understand the binding and inhibition mechanism of these variants.This article is protected by copyright. All rights reserved.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display

Hosseini,

Kumar,

Hedin

et al. 2023

Protein Science

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“…Use of a yeast surface display platform to anchor recombinant proteins to yeast cell wall has been widely adapted in the biomedical field for antibody and antimicrobial peptide engineering, protein stability, protein–protein interaction, and enzyme metabolic studies ( Cherf and Cochran, 2015 ; Wen et al, 2018 ; Medina-Cucurella et al, 2019 ; Lau et al, 2021 ; Luo et al, 2021 ; Fiebig et al, 2022 ; Kuroda and Ueda, 2022 ; Raeeszadeh-Sarmazdeh and Boder, 2022 ; Teymennet-Ramirez et al, 2022 ). A common yeast display system involves fusing recombinant proteins to the a-agglutinin mating protein Aga2p and expressing extracellularly in yeast that secrete the ß 1,6 glucan-anchored Aga1p domain of a-agglutinin to form an Aga1p–Aga2p complex via two disulfide bonds ( Wang et al, 2005 ; Cherf and Cochran, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations

Liu

Patel

et al. 2023

Front. Bioeng. Biotechnol.

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Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and use of antibiotics. Recent emphasis on antibiotic stewardship has prompted new research into the development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, which inhibited Limosilactobacillus fermentum strains in corn mash fermentation. Here, Saccharomyces cerevisiae EBY100 was used to anchor recombinant LysKB317 using cell surface display with the a-agglutinin proteins Aga1p–Aga2p. Immunostaining and confocal fluorescence were used for localization of the extracellular interface of the cells. Yeast surface-expressed endolysin demonstrated an 83.8% decrease in bacterial cell counts compared to a 9.5% decrease in control yeast. Recombinant S. cerevisiae expressing LysKB317 used for small-scale corn mash fermentation, when infected with L. fermentum, could proactively control bacterial infection for 72 h with at least 1-log fold reduction. Analysis of fermentation products showed improved ethanol concentrations from 3.4% to at least 5.9% compared to the infection-only control and reduced levels of lactic and acetic acid from 34.7 mM to 13.8 mM and 25.5 mM to 18.1 mM, respectively. In an optimized yeast surface display system, proactive treatment of bacterial contaminants by endolysin LysKB317 can improve fermentation efficiency in the presence of L. fermentum contamination.

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“…The use of a yeast surface display platform to anchor recombinant proteins to the cell wall of yeast has been widely adapted in the biomedical eld for antibody and antimicrobial peptide engineering, protein stability, protein-protein interaction, and enzyme metabolic studies [21][22][23][24][25][26][27][28][29]. A common yeast display system involves fusing recombinant proteins to the α-agglutinin mating protein Aga2p and expressing extracellularly in yeast that secrete the β 1,6 glucan-anchored Aga1p domain of α-agglutinin to form an Aga1p-Aga2p complex via two disul de bonds [21,30].…”

Section: Introductionmentioning

confidence: 99%

Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations

Liu

Patel

et al. 2022

Preprint

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Background Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and the use of antibiotics. Recent emphasis on antibiotic stewardship has prompted new research into development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, that inhibited Lactobacillus strains in corn mash fermentation. Here, Saccharomyces cerevisiae EBY100 was used to anchor recombinant LysKB317 using cell surface display with the α-agglutinin proteins Aga1p-Aga2p. Results Immunostaining and confocal fluorescence were used for localization of the extracellular interface of the cells. Yeast surface expressed endolysin demonstrated an 83.8% decrease in bacterial cell counts compared to 9.5% decrease in control yeast. Recombinant S. cerevisiae expressing LysKB317 used for small-scale corn mash fermentation, when infected with Limosilactobacillus fermentum, showed the ability to proactively control bacterial infection for 72 hours with at least 1-log fold reduction. HPLC analysis of fermentation products showed improved ethanol concentration from 3.4% to at least 5.9% compared to infection-only control and reduced levels of lactic and acetic acid from 34.7 mM to 13.8 mM and 25.5 mM to 18.1 mM in that order. Conclusion In an optimized yeast surface display system, proactive treatment of bacterial contaminants by endolysin LysKB317 can improve fermentation efficiency in the presence of L. fermentum contamination.

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Yeast Surface Display: New Opportunities for a Time-Tested Protein Engineering System

Cited by 11 publications

References 136 publications

Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display

Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display

Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations

Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations

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