Secretory Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Kluyveromyces lactis and Characterization of Its Biological Activity

Kuduğ, Hülya; Tayhan, Seçil Erden; Gökçe, İ̇sa

doi:10.1007/s10989-021-10227-7

Cited by 5 publications

(2 citation statements)

References 64 publications

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“…Recombinant Kluyveromyces lactis GG799 (New England Biolabs, Massachusetts, USA) was prepared by cloning the human VEGF 165 cDNA (GenBank accession no. AF486837.1) in Xho I and EcoR I sites of the pKLAC2 vector under the control of the LAC4 promoter of the reported study [ 38 ]. The recombinant strain was cultured in YCB plates (3% 1 M Tris–HCl pH 7.4, 1.17% YCB, and 2% agar) containing 5 mM acetamide and maintained at – 80 °C as glycerol stock.…”

Section: Methodsmentioning

confidence: 99%

A colorimetric immunoassay for the detection of human vascular endothelial growth factor 165 (VEGF165) based on anti-VEGF-iron oxide nanoparticle conjugation

Ceylan,

Kırbay,

Yazgan

et al. 2024

Microchim Acta

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Vascular endothelial growth factor (VEGF) is an indispensable element in many physiological processes, while alterations in its level in the circulating system are signs of pathology-associated diseases. Therefore, its precise and selective detection is critical for clinical applications to monitor the progression of the pathology. In this study, an optical immunoassay biosensor was developed as a model study for detecting recombinant VEGF165. The VEGF165 sample was purified from recombinant Kluyveromyces lactis GG799 yeast cells. Indirect ELISA was used during the detection, wherein iron oxide nanoparticles (FeNPs) were utilized to obtain optical signals. The FeNPs were synthesized in the presence of lactose p-amino benzoic acid (LpAB). VEGF165 antibody was conjugated to the LpAB-FeNPs through EDC/NHS chemistry to convert the iron oxide nanoparticles into VEGF165 specific probes. The specificity of the prepared system was tested in the presence of potential serum-based interferents (i.e., glucose, urea, insulin, C-reactive protein, and serum amyloid A), and validation studies were performed in a simulated serum sample. The proposed immunoassay showed a wide detection range (0.5 to 100 ng/mL) with a detection limit of 0.29 ng/mL. These results show that the developed assay could offer a sensitive, simple, specific, reliable, and high-throughput detection platform that can be used in the clinical diagnostics of VEGF. Graphical Abstract

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Section: Methodsmentioning

confidence: 99%

A colorimetric immunoassay for the detection of human vascular endothelial growth factor 165 (VEGF165) based on anti-VEGF-iron oxide nanoparticle conjugation

Ceylan,

Kırbay,

Yazgan

et al. 2024

Microchim Acta

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“…Recombinant K. lactis strain GG799 (New England BioLabs, Massachusetts, USA) was previously constructed by inserting the human VEGF165 cDNA (GenBank accession no. AF486837.1) into the XhoI and EcoRI sites of the pKLAC2 vector under the control of the LAC4 promoter [25]. The recombinant strain was cultivated on YCB plates (3% 1 M Tris-HCl pH 7.4, 1.17% YCB, and 2% agar) containing 5 mM acetamide and preserved at −80 °C as a glycerol stock.…”

Section: Reagents Strains and Growth Mediummentioning

confidence: 99%

Optimization of Medium Components for Enhanced Expression of Recombinant Human Vascular Endothelial Growth Factor (VEGF165) in Kluyveromyces lactis GG799

Kuduğ Ceylan,

Meydan,

Nalbantsoy

et al. 2024

Türk Doğa Ve Fen Dergisi

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Vasküler endotelyal büyüme faktörü (VEGF165), anjiyogenezdeki rolünden dolayı çeşitli fizyolojik süreçler ve kanser gibi patolojik hastalıklar ile ilişkili önemli bir biyobelirteçtir. Bu nedenle, yüksek verimde rekombinant VEGF165 elde etmek için etkili bir yöntem gereklidir. Bu çalışmada, Kluyveromyces lactis (K. lactis) GG799 hücrelerinde rekombinant VEGF165 ekspresyonunu artırmak amacıyla büyüme ortamının optimize edilmesi amaçlanmıştır. Farklı ortam bileşenlerinin taranması sonucunda galaktoz, tripton ve maya ekstraktı en etkili bileşenler olarak belirlenmiştir. Box-Behnken tasarımı kullanılarak seçilen ortam bileşenleri optimize edilmiş ve biyokütle konsantrasyonunda %31'lik kayda değer bir artış sağlanmıştır. 45 g L-1 galaktoz, 30 g L-1 tripton ve 5 g L-1 maya ekstraktı içeren optimize ortam ile 0,26 gDCW L-1 h-1 biyokütle üretkenliğine (P) ulaşılmıştır. Ayrıca VEGF165 ekspresyonu, optimize edilmemiş ortama kıyasla 1,27 kat artışla 1024,09 μg L-1 değerine ulaşmıştır. Elde edilen sonuçlar, önerilen optimize edilmiş ortamın K. lactis'te diğer rekombinant proteinlerin üretimine yönelik proses geliştirme stratejisi olarak bir model olabileceğini göstermektedir.

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Enhanced Biomass Production of Recombinant Pfu DNA Polymerase Producer Escherichia coli BL21(DE3) by Optimization of Induction Variables Using Response Surface Methodology

Kuduğ

2023

Protein J

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Secretory Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Kluyveromyces lactis and Characterization of Its Biological Activity

Cited by 5 publications

References 64 publications

A colorimetric immunoassay for the detection of human vascular endothelial growth factor 165 (VEGF165) based on anti-VEGF-iron oxide nanoparticle conjugation

A colorimetric immunoassay for the detection of human vascular endothelial growth factor 165 (VEGF165) based on anti-VEGF-iron oxide nanoparticle conjugation

Optimization of Medium Components for Enhanced Expression of Recombinant Human Vascular Endothelial Growth Factor (VEGF165) in Kluyveromyces lactis GG799

Enhanced Biomass Production of Recombinant Pfu DNA Polymerase Producer Escherichia coli BL21(DE3) by Optimization of Induction Variables Using Response Surface Methodology

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