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

Leow HC, Fischer K, Leow YC, Braet K, Cheng Q, McCarthy J

Prep Biochem Biotechnol, 2019;49(4):315-327.
PMID: 30767708 DOI: 10.1080/10826068.2019.1566145

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.
Fulltext Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library

Leow CH, Fischer K, Leow CY, Braet K, Cheng Q, McCarthy J

Malar J, 2018 Oct 24;17(1):383.
PMID: 30355309 DOI: 10.1186/s12936-018-2531-y

BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.
METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.

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