Structural Characteristics of Hen Egg Ovalbumin Expressed in Yeast<i>Pichia pastoris</i>

Ito, Kazunari; Matsudomi, Naotoshi

doi:10.1271/bbb.69.755

Cited by 18 publications

(6 citation statements)

References 26 publications

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“…We used both circular dichroism (CD) and intrinsic fluorescence (Ex280, Em330/350) to measure the changes in secondary and tertiary elements, respectively. Our thermal stability data for ovalbumin ( Figure 2B & 2C ) was similar to previously published data (48) given the difference in buffers ( Table 1 & 2 ). For both DSF and DSCD, Cdt1 followed a simple two-state unfolding process.…”

Section: Resultssupporting

confidence: 89%

SAXS/MC studies of the mixed-folded protein Cdt1 reveal monomeric, folded over conformations

Smith,

Chakravarthy,

Rahi

et al. 2024

Preprint

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Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, “moonlighting” role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the proteinin silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent “folded over” conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.

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Section: Resultssupporting

confidence: 89%

SAXS/MC studies of the mixed-folded protein Cdt1 reveal monomeric, folded over conformations

Smith,

Chakravarthy,

Rahi

et al. 2024

Preprint

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“…Moreover, different biological functions were evidenced for these OVA glycosylation sites. N 293 , as an example, was proven to be vital for the secretion and conformation of OVA in the yeast expression system, whereas the N 311 glycosylation site had no such effect. , During incubation of fertilized eggs, inconsistent variations were observed for these OVA glycosylation sites. In comparison of D12 to D0, five glycosylation sites were upregulated, while six glycosylation sites were downregulated, with a fold change of over 1.2-fold (D12/D0) (Table S1 of the Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Integrated Proteomic and N-Glycoproteomic Analyses of Chicken Egg during Embryonic Development

Zhu

Qiu

Sun

et al. 2019

J. Agric. Food Chem.

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To better appreciate the alterations of egg proteins and their modifications during embryonic development, a comparative and quantitative study was performed aimed at chicken egg white and yolk proteome and N-glycoproteome after 12 days of incubation using tandem mass tag (TMT)-labeling technology in conjunction with reversed-phase high-performance liquid chromatography (RP-HPLC). A total of 334 unique N-glycosite-containing peptides from 153 N-glycoproteins were identified, of which 82 N-glycosite-containing peptides showed significant changes after 12 days of incubation. The varied proteome was mainly involved with antibacterial, ionic binding, cell proliferation, and embryonic development, while the different degrading and/or absorbing priorities of egg proteins were proposed. This study provides substantial insight into the effects of N-glycoprotein variations on the utilization of egg proteins by chicken embryo during incubation.

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“…Ovalbumin was expressed with Ko. phaffii , resulting in two molecular species with similar molecular weight, 45 and 47 kDa, likely representing different glycoforms (Ito & Matsudomi, 2005). Approximately 10 mg/ml was recovered; however, expression was performed using a small volume (3 ml) and the constitutive GAP promoter.…”

Section: Pcag In the Public Domainmentioning

confidence: 99%

“…Recombinant proteins denatured at ∼2.5°C lower than native ovalbumin, however, due to the lack of phosphorylation. Dephosphorylated native ovalbumin had a melting temperature of 75.1°C, compared to 74.8–74.9°C in the different recombinant proteins (Ito & Matsudomi, 2005). Removal of the Asn292 glycosylation site abolished secretion, even with the other native glycosylation site intact (Asn311), or with new ones added (Ser168Asn or Ser236Asn) (Ito, Seri, et al., 2007; Ito, Ishimaru, et al., 2007).…”

Section: Pcag In the Public Domainmentioning

confidence: 99%

Precision cellular agriculture: The future role of recombinantly expressed protein as food

Dupuis

Cheung

Newman

et al. 2022

Comp Rev Food Sci Food Safe

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Cellular agriculture is a rapidly emerging field, within which cultured meat has attracted the majority of media attention in recent years. An equally promising area of cellular agriculture, and one that has produced far more actual food ingredients that have been incorporated into commercially available products, is the use of cellular hosts to produce soluble proteins, herein referred to as precision cellular agriculture (PCAg). In PCAg, specific animal‐ or plant‐sourced proteins are expressed recombinantly in unicellular hosts—the majority of which are yeast—and harvested for food use. The numerous advantages of PCAg over traditional agriculture, including a smaller carbon footprint and more consistent products, have led to extensive research on its utility. This review is the first to survey proteins currently being expressed using PCAg for food purposes. A growing number of viable expression hosts and recent advances for increased protein yields and process optimization have led to its application for producing milk, egg, and muscle proteins; plant hemoglobin; sweet‐tasting plant proteins; and ice‐binding proteins. Current knowledge gaps present research opportunities for optimizing expression hosts, tailoring posttranslational modifications, and expanding the scope of proteins produced. Considerations for the expansion of PCAg and its implications on food regulation, society, ethics, and the environment are also discussed. Considering the current trajectory of PCAg, food proteins from any biological source can likely be expressed recombinantly and used as purified food ingredients to create novel and tailored food products.

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Structural Characteristics of Hen Egg Ovalbumin Expressed in YeastPichia pastoris

Cited by 18 publications

References 26 publications

SAXS/MC studies of the mixed-folded protein Cdt1 reveal monomeric, folded over conformations

SAXS/MC studies of the mixed-folded protein Cdt1 reveal monomeric, folded over conformations

Integrated Proteomic and N-Glycoproteomic Analyses of Chicken Egg during Embryonic Development

Precision cellular agriculture: The future role of recombinantly expressed protein as food

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