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Molecular Biotechnology

, Volume 60, Issue 8, pp 585–594 | Cite as

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in Escherichia coli

  • Rufus Vinod Munawar Samuel
  • Syeda Yumna Farrukh
  • Sadia Rehmat
  • Muhammad Umair Hanif
  • Syed Shoaib Ahmed
  • Syed Ghulam Musharraf
  • Faiza Gul Durrani
  • Mahjabeen Saleem
  • Roquyya Gul
Original Paper
First Online:

Abstract

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon + and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~ 25.5% and ~ 6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.

Keywords

HrVEGF-A121 hSUMO3 Rosetta-gami B (DE3) Fast protein liquid chromatography ESI-MS MTT 
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Notes

Author Contributions

All the authors have seen and approved the manuscript and performed their responsibilities in designing the project, experimental work, and manuscript write up.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no financial or commercial conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

12033_2018_94_MOESM1_ESM.docx (377 kb)
Supplementary material 1 (DOCX 377 KB)

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Rufus Vinod Munawar Samuel
    • 1
  • Syeda Yumna Farrukh
    • 1
  • Sadia Rehmat
    • 1
  • Muhammad Umair Hanif
    • 1
  • Syed Shoaib Ahmed
    • 1
  • Syed Ghulam Musharraf
    • 2
  • Faiza Gul Durrani
    • 3
  • Mahjabeen Saleem
    • 4
  • Roquyya Gul
    • 1
  1. 1.Institute of Molecular Biology and Biotechnology/Centre for Research in Molecular MedicineThe University of LahoreLahorePakistan
  2. 2.Dr. Panjwani Center for Molecular Medicine and Drug Research/International Center for Chemical and Biological SciencesUniversity of KarachiKarachiPakistan
  3. 3.School of Biological SciencesUniversity of the PunjabLahorePakistan
  4. 4.Institute of Biochemistry and BiotechnologyUniversity of the PunjabLahorePakistan

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