Leow Herng C, Fischer Katja, Leow Yee C, Braet Katleen, Cheng Qin, McCarthy James
a Institute for Research in Molecular Medicine (INFORMM) , Universiti Sains Malaysia , Penang , Malaysia.
b Clinical Tropical Medicine Department, QIMR Berghofer Medical Research Institute , Brisbane , Australia.
Prep Biochem Biotechnol. 2019;49(4):315-327. doi: 10.1080/10826068.2019.1566145. Epub 2019 Feb 15.
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.
已知鲨鱼可变新抗原受体（VNARs）具有出色的热稳定性，其长互补决定区3（CDR3）使其能够穿透到抗原的裂隙区域。VNAR中所含半胱氨酸（Cys）残基的数量比传统抗体中的多，这就要求在环间或环内区域形成二硫键以与靶蛋白抗原相互作用。因此，选择合适的表达系统对于确保功能性VNAR蛋白产生的溶解性和正确折叠很重要。与高等生物不同，大肠杆菌（E. coli）中进行蛋白质翻译后修饰的机制不太完善。为了克服这种情况，构建了一种与DsbA信号肽融合的pDSB - 28Y载体，用于周质H8VNAR的产生。尽管周质蛋白的产量（62 µg/mL）低于从pET - 28a载体获得的胞质蛋白（468 µg/mL），但在吸光度方面它表现出比胞质蛋白更好的性能。然而，在本实验中这些读数仍低于阳性对照小鼠单克隆抗体（mAb）C1 - 13。因此，需要进一步研究以提高所选重组VNAR对疟疾生物标志物的结合亲和力。
