title: High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.
abstract: Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, 13164, Tehran, Iran.
authors.forename: Najmeh
authors.initials: N
authors.lastname: Zarei
authors.forename: Behrouz
authors.initials: B
authors.lastname: Vaziri
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Shokrgozar
authors.forename: Reza
authors.initials: R
authors.lastname: Mahdian
authors.forename: Ramin
authors.initials: R
authors.lastname: Fazel
authors.forename: Vahid
authors.initials: V
authors.lastname: Khalaj
mesh_terms: D003062:Codon / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D000431:Ethanol / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000037:antagonists & inhibitors*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors
chemical_list: C497867:CD22 protein, human; D003062:Codon; D003470:Culture Media; D011994:Recombinant Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; D057127:Single-Chain Antibodies; D000431:Ethanol