Cytoplasmic and periplasmic expression of recombinant shark VNAR antibody in Escherichia coli

Abstract

Shark variable new antigen receptors (VNARs) are known to possess excellent heat-stability, and the long complementarity determining region 3 (CDR3) has permitted it to penetrate into the cleft region of antigens. The number of cysteine (Cys) residues contained within VNAR is greater than in conventional antibodies, entailing disulfide bond formation in both the inter- or intra-loop regions is required for interactions with the target protein antigens. Therefore, the selection of a suitable expression system is important to ensure the solubility and correct folding of functional VNAR protein production. Unlike higher organisms, the machinery for effecting posttranslational modifications of proteins in Escherichia coli (E. coli) are less sophisticated. To overcome this circumstance, a pDSB-28Y vector fusion with DsbA signal peptide was engineered for periplasmic H8VNAR production. Despite the periplasmic proteins showing a lower yield (62 µg/mL) than cytosolic proteins (468 µg/mL) that is obtained from pET-28a vector, it has demonstrated better performance than that of a cytosolic protein in terms of absorbance. However, these readings were still inferior to that of positive control mouse monoclonal antibody (mAb) C1-13 in this experiment. Therefore, further investigation is required to improve the binding affinity of selected recombinant VNAR towards malaria biomarkers.

Keywords:

• Antibody
• cytosolic
• disulfide bonds
• E. coli expression system
• periplasmic
• shark VNAR
• single domain antibody

Acknowledgments

We would like to thank Dr Martin Bubb, National Products Institute, South Africa for providing mAb C1-13. Also special thanks to Dr. Mark Pearson from James Cook University, Australia for providing the pSSPET vector. The opinions expressed herein are those of the authors and do not necessarily reflect those of the Australian Defense Force Joint Health Command.

Additional information

Funding

We appreciate the funding sponsored by Malaysian Government and University Sains Malaysia, including Malaysian Ministry of Higher Education the Higher Institutions Centre of Excellence Program (Grant no.: 311/CIPPM/4401005), RUI Grant no.: RU(1001/CIPPM/8011050) and USM Short Term (Grant no.: 304/CIPPM/6313191) to complete this project.