Beta-Lactoglobulin as a Model Food Protein: How to Promote, Prevent, and Exploit Its Unfolding Processes

Barbiroli, Alberto; Iametti, Stefania; Bonomi, Francesco

doi:10.3390/molecules27031131

Cited by 16 publications

(6 citation statements)

References 60 publications

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“…However, some proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins act as allergens to susceptible individuals 2 . Among these proteins, BLG is a major allergen in the milk of farm animals, including buffaloes 3 . Traditional efforts to reduce milk allergenicity involve processing methods like pasteurization or ultra-high temperature treatment to denature milk proteins 2 .…”

Section: Introductionmentioning

confidence: 99%

CRISPR-mediated editing of β-lactoglobulin (BLG) gene in buffalo

Tara,

Singh,

Gautam

et al. 2024

Sci Rep

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Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (−/−) heterozygous, bi-allelic (−/−) homozygous, and mono-allelic (−/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (−/−) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

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Section: Introductionmentioning

confidence: 99%

CRISPR-mediated editing of β-lactoglobulin (BLG) gene in buffalo

Tara,

Singh,

Gautam

et al. 2024

Sci Rep

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“…Despite these encouraging interfacial mechanical properties, BSA and BLG remain inherently globular proteins playing roles in molecular transport and lipid stabilization in physiological fluids, − with little relevance to ECM signaling and the promotion of cell adhesion. To confer cell adhesion to these scaffold proteins, we coupled an RGD peptide to the surface of protein nanosheet-stabilized droplets.…”

Section: Resultsmentioning

confidence: 99%

Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilization of Bioemulsions Enabling Adherent Cell Expansion

2023

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Bioemulsions are attractive platforms for the scalable expansion of adherent cells and stem cells. In these systems, cell adhesion is enabled by the assembly of protein nanosheets that display high interfacial shear moduli and elasticity. However, to date, most successful systems reported to support cell adhesion at liquid substrates have been based on coassemblies of protein and reactive cosurfactants, which limit the translation of bioemulsions. In this report, we describe the design of protein nanosheets based on two globular proteins, bovine serum albumin (BSA) and β-lactoglobulin (BLG), biofunctionalized with RGDSP peptides to enable cell adhesion. The interfacial mechanics of BSA and BLG assemblies at fluorinated liquid-water interfaces is studied by interfacial shear rheology, with and without cosurfactant acyl chloride. Conformational changes associated with globular protein assembly are studied by circular dichroism and protein densities at fluorinated interfaces are evaluated via surface plasmon resonance. Biofunctionalization mediated by sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is studied by fluorescence microscopy. On the basis of the relatively high elasticities observed in the case of BLG nanosheets, even in the absence of cosurfactant, the adhesion and proliferation of mesenchymal stem cells and human embryonic kidney (HEK) cells on bioemulsions stabilized by RGD-functionalized protein nanosheets is studied. To account for the high cell spreading and proliferation observed at these interfaces, despite initial moderate interfacial elasticities, the deposition of fibronectin fibers at the surface of corresponding microdroplets is characterized by immunostaining and confocal microscopy. These results demonstrate the feasibility of achieving high cell proliferation on bioemulsions with protein nanosheets assembled without cosurfactants and establish strategies for rational design of scaffolding proteins enabling the stabilization of interfaces with strong shear mechanics and elasticity, as well as bioactive and cell adhesive properties. Such protein nanosheets and bioemulsions are proposed to enable the development of new generations of bioreactors for the scale up of cell manufacturing.

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“…Despite these encouraging interfacial mechanical properties, BSA and BLG remain inherently globular proteins playing roles in molecular transport and lipid stabilization in physiological fluids [58][59][60] , with little relevance to ECM signaling and the promotion of cell adhesion. To confer cell adhesion to these scaffold proteins, we coupled an RGD peptide to the surface of protein nanosheet-stabilized droplets.…”

Section: Resultsmentioning

confidence: 99%

Engineering of Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilisation of Bioemulsions Enabling Adherent Cell Expansion

Chrysanthou

Bosch‐Fortea

Gautrot

2022

Preprint

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Bioemulsions are attractive platforms for the scalable expansion of adherent cells and stem cells. In these systems, cell adhesion is enabled by the assembly of protein nanosheets that display high interfacial shear moduli and elasticity. However, to date, most successful systems reported to support cell adhesion at liquid substrates have been based on co-assemblies of protein and reactive co-surfactants, which limit the translation of bioemulsions. In this report, we describe the design of protein nanosheets based on two globular proteins, bovine serum albumin (BSA) and β-lactoglobulin (BLG), biofunctionalised with RGDSP peptides to enable cell adhesion. The interfacial mechanics of BSA and BLG assemblies at fluorinated liquid-water interfaces is studied by interfacial shear rheology, with and without co-surfactant acyl chloride. Conformational changes associated with globular protein assembly are studied by circular dichroism and protein densities at fluorinated interfaces are evaluated via surface plasmon resonance. Biofunctionalisation mediated by sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is studied by fluorescence microscopy. On the basis of the relatively high elasticities observed in the case of BLG nanosheets, even in the absence of co-surfactant, the adhesion and proliferation of mesenchymal stem cells and human embryonic kidney (HEK) cells on bioemulsions stabilized by RGD-functionalized protein nanosheets is studied. To account for the high cell spreading and proliferation observed at these interfaces, despite initial moderate interfacial elasticities, the deposition of fibronectin fibers at the surface of corresponding microdroplets is characterized by immunostaining and confocal microscopy. These results demonstrate the feasibility of achieving high cell proliferation on bioemulsions with protein nanosheets assembled without co-surfactants and establish strategies for rational design of scaffolding proteins enabling the stabilization of interfaces with strong shear mechanics and elasticity, as well as bioactive and cell adhesive properties. Such protein nanosheets and bioemulsions are proposed to enable the development of new generations of bioreactors for the scale up of cell manufacturing.

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Beta-Lactoglobulin as a Model Food Protein: How to Promote, Prevent, and Exploit Its Unfolding Processes

Cited by 16 publications

References 60 publications

CRISPR-mediated editing of β-lactoglobulin (BLG) gene in buffalo

CRISPR-mediated editing of β-lactoglobulin (BLG) gene in buffalo

Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilization of Bioemulsions Enabling Adherent Cell Expansion

Engineering of Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilisation of Bioemulsions Enabling Adherent Cell Expansion

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