Secreted Expression of an Active Human Interferon-Beta (HuIFNβ) in Kluyveromyces Lactis

Abstract

Interferon-beta (IFNβ) is a cytokine involved in the antiviral, anti-proliferative and immunomodulatory responses of cells, and a potent drug for multiple sclerosis. Human interferon beta (HuIFNβ) gene fused with the glucoamylase signal sequence in the N-terminus and 6 His Tag in the C-terminus was cloned into pKlac1 vector and introduced in Kluyveromyces lactis to allow secreted expression and one-step purification of the protein. Recombinant yeast transformant with the highest level ofHuIFNβ production was identified, and this secreted up to 1 mg/L of the cytokine after 72 h of incubation. Glycosylated and non-glycosylated forms of the cytokine were elaborated by the yeast, the latter in higher quantities. His tag of the protein allowed easy one-step purification by nickel-nitriloacetic acid affinity chromatography, yielding close to 100% purity. SDS-PAGE, western blot andMALDI-TOF-TOF confirmed the identity of the protein. The biological activity of the recombinant HuIFNβ was confirmed by its anti cell proliferative activity on HeLa cells. Expression ofHuIFNβ in K. lactis is advantageous since it is a very safe organism to produce proteins for therapeutic applications, allows glycosylation and offers a cost effective method for large scale production as it can be grown in cheap carbon sources