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Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain

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Abstract

Vascular endothelial growth factor A165 (VEGF-A165) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A165 receptor-binding domain (VEGFA165-RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA165-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA165-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA165-RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35–180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA165-RBD. 25 ng sKDR1-3 and 20 ng VEGFA165-RBD showed strong binding. sKDR1-3 bound to VEGFA165-RBD and VEGF-A165 with dissociation constants of 0.148 and 0.2 nM, respectively. 4–10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA165-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.

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Data Availability

The data that support the findings of this study are available on request from the corresponding author.

Abbreviations

VEGF:

Vascular endothelial growth factor

VEGFR-2:

VEGF receptor 2

VEGFA165-RBD:

VEGF-A165 receptor binding domain

VEGFA165-HBD:

VEGF-A165 heparin binding domain

KDR:

Kinase domain receptor

YPD:

Yeast Extract-Peptone-Dextrose Medium

YNB:

Yeast nitrogen base

Avastin:

An anti-VEGF-A165 monoclonal antibody

AOX1 :

alcohol oxidase I

ADE2:

Auxotrophic marker

amp:

Ampicillin

AmpR:

Ampicillin resistance

A280 :

Absorbance at 280 nm

BSA:

Bovine serum albumin

PBS:

Phosphate buffered saline

βME:

β-Mercaptoethanol

BMGY:

Buffered glycerol complex medium

BMMY:

Buffered methanol-complex medium

CYC1 TT:

Transcription termination of CYC1

DTT:

Dithiothreitol

ELISA:

Enzyme-linked immunosorbent assay

E. coli :

Escherichia coli

EDTA:

Ethylenediaminetetraacetic acid

G4S linker:

Gly4Ser

HGF:

Hepatocyte growth factor

His:

Histidine

HRP:

Horse radish peroxidase

HUVE:

Human umbilical vein endothelial cells

Ig:

Immunoglobulin-like

IgG:

Immunoglobulin class G

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

IC50 :

Half-maximum inhibitory concentration

Kaff :

Affinity constant

KD:

Dissociation constant

LBD:

Ligand binding domain

LB:

Luria–Bertani

Ni–NTA:

Nickel-nitrilotriaceticacid

NP:

Normal physiological proteins

nM:

Nanomolar

ms:

Millisecond

OD:

Optical density

OD600 :

Optical density at 600 nm

OD450 :

Optical density at 400 nm

PBS:

Phosphate buffered saline

PADE2 HC:

ADE2 promoter

Pad:

Pichia Adenine Dropout

PBST:

PBS containing tween 20

PAOX1 :

AOX1 promoter

PCR:

Polymerase chain reaction

PDB:

Protein data bank

pUC ori:

PUC origin of replication

PIGF:

Placental growth factor

RBD:

Receptor-binding domain

SDS-PAGE:

Sodium dodecyl sulfatepolyacrylamide gel electrophoresis

sKDR1-3:

Solube KDR domains 1–3

TB:

Terrific broth

TMB:

Tetramentylbenzidine

TBST:

TBS containing tween 20 and BSA

TRP2 gene:

Integration locus

TBS:

Tris-buffered saline

SXA :

β-D-Xylosidase/α-ʟ-arabinofuranosidase from the ruminal anaerobic bacterium Selenomonas ruminantium

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Acknowledgements

The authors thank the universities of Kharazmi, Maragheh, and Tarbiat Modares of Iran.

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Authors and Affiliations

  1. Department of Cell and Molecular Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

    Zahra Fathi & Masoud Mashhadi Akbar Boojar

  2. Department of Biology, Faculty of Sciences, University of Maragheh, Maragheh, Iran

    Zahra Fathi & Zohreh Jahanafrooz

  3. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Reza H. Sajedi

  4. Gene Transfer Pioneers (GTP) Research Group, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Ehsan Dehnavi

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  1. Zahra Fathi
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Contributions

ZJ, ZF, MMAB, and RHS conceived the idea and developed the theory. ZF and ED performed the experiments, verified the methods, supervised the findings, and analyzed the data. ZJ and ZF designed the manuscript and provided critical feedback to help shape the manuscript. All authors approved the final version.

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Correspondence to Zohreh Jahanafrooz.

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Fathi, Z., Boojar, M.M.A., Sajedi, R.H. et al. Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain. Mol Biotechnol (2024). https://doi.org/10.1007/s12033-024-01057-1

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