Engineering and characterization of a recombinant human interferon alpha-2b fused with apolipoprotein A-I possessing prolonged action

Recombinant human interferon alpha-2b (rIFN) is widely used for the treatment of both viral and oncological diseases. The short half-life and toxicity of rIFN are a serious limitation for the use of rIFN in therapy. In this regard, the development of a less toxic rIFN with a longer half-life is an important goal of medical biotechnology. Currently, there are two main modifications of long-acting rIFN - pegylated rIFN and rIFN genetically fused with albumin. The use of human apolipoprotein A-I (apoA-I) as a protector protein seems also promising, since apoA-I has a long half-life in the body, is not immunogenic, is capable of natural formation of lipoprotein complexes, and allows improve the pharmacokinetics of therapeutic proteins fused with it.

In this work, a recombinant strain of Pichia pastoris X33, producing a chimeric protein consist of rIFN, fused with human apoA-I through a flexible linker, was obtained. The IFN and apoA-I genes were optimized for expression in P. pastoris. The cultivation of a yeast strain producing a chimeric cytokine was carried out in an orbital shaker. The protein yield was 30 mg/L. The chimera was purified by reversed phase chromatography. The purity of the final preparation of the chimera was about 90%. The primary structure of the chimera was confirmed by MALDI-TOF. The chimeric cytokine exhibited high specific antiviral activity comparable to that of rIFN and equal to 1.6 × 10⁸ IU/mg. The chimera showed a 1.8-fold longer half-life in comparison with rIFN аfter a single subcutaneous injection in mice.