Functional recombinant extra membrane loop of human CD20, an alternative of the full length CD20 antigen

Abstract

Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20.

Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein.

Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems.

Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies.

Keywords: E. Coli; CD20; Thioredoxin.

Fig. 1

Gel electrophoresis of PCR product and colony PCR. The 141-bp exCD20 coding sequence was amplified by PCR and 2 µl sample of PCR product was run on a 1.5% agarose gel (A). This sequence was cloned into pET32a(+) expression vector and the cloning was confirmed by colony PCR. The amplified product was visualized around 720 bp on a 1% agarose gel (582 bp, from T7 promoter binding site to BamHI cut site + 141 bp, the size of insert) (B).

Fig. 2

Chromatogram of size exclusion chromatography. The sample was loaded onto a Superdex 75 column. Two overlapped main protein peaks in addition to some small protein peaks (peaks 1 and 2) were separated. A conductivity peak corresponding to the imidazole was obtained at the end of size exclusion chromatography experiment.

Fig. 3

SDS-PAGE and Western-blot analyses of size exclusion chromatography (SEC) column purified samples. One µg from peaks 1 and 2 of chromatography-purified SEC fraction was subjected to SDS-PAGE (10% acrylamide gel) under reducing conditions (A). Separated proteins were then transferred to the nitrocellulose membrane and stained by a mouse anti-CD20 peptide antibody and anti-mouse alkaline phosphatase-conjugated antibody as primary and secondary antibodies, respectively (B).