Novel recombinant feline interferon carrying N-glycans with reduced allergy risk produced by a transgenic silkworm system

Sachi Minagawa,Yuzuru Nakaso,Takenori Igarashi,Masahiro Tomita,Yoshio Miura,Hideyo Yasuda,Satoshi Sekiguchi

doi:10.1186/s12917-018-1584-z

Sachi Minagawa, Yuzuru Nakaso + Show 5 more

Open Access

https://doi.org/10.1186/s12917-018-1584-z

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Abstract

BackgroundThe generation of recombinant proteins for commercialisation must be cost-effective. Despite the cost-effective production of recombinant feline interferon (rFeIFN) by a baculovirus expression system, this rFeIFN carries insect-type N-glycans, with core α 1,3 fucosyl residues that act as potential allergens. An alternative method of production may yield recombinant glycoproteins with reduced antigenicity.ResultsA cDNA clone encoding the fifteenth subtype of FeIFN-α (FeIFN-α15) was isolated from a Japanese domestic cat. This clone encoded a protein of 189 amino acids with a molecular mass of 21.1 kDa. The rFeIFN-α15 was expressed using a transgenic silkworm system, which was expected to yield an N-glycan structure with reduced antigenicity compared with the protein produced by the baculovirus system. The resulting rFeIFN-α15 accumulated in the sericin layer of silk fibres and was easily extracted and purified by column chromatography. The N-terminal amino acid sequence of purified rFeIFN-α15 was identical to the mature form of natural sequence. Moreover, its N-glycans did not include detectable core α 1,3 fucosyl residues. Its anti-vesicular stomatitis virus activity (2.6 × 108 units/mg protein) was comparable to that of the baculovirus-expressed rFeIFN.ConclusionsThe lower allergy risk of rFeIFN produced by the transgenic silkworm system than by the baculovirus expression system is due to the former lacking core α 1,3 fucosyl residues in its N-glycans. The rFeIFN-α15 produced by the transgenic silkworm system may be a prospective candidate for the next generation of rFeIFN in veterinary medicine.

Highlights

The generation of recombinant proteins for commercialisation must be cost-effective
Using a cDNA clone encoding a newly discovered Feline interferon (FeIFN) α subtype, FeIFN-α15, isolated from the blood of a Japanese domestic cat, this study evaluated the properties of recombinant feline interferon (rFeIFN) produced by the transgenic silkworm system
Isolation of a clone of FeIFN Total mRNA was isolated from leucocytes of a domestic cat, and a FeIFN clone was isolated by Polymerase chain reaction (PCR) using primers designed from the sequences of FeIFN 7 and FeIFN 8

Summary

Introduction

The generation of recombinant proteins for commercialisation must be cost-effective. Despite the cost-effective production of recombinant feline interferon (rFeIFN) by a baculovirus expression system, this rFeIFN carries insect-type N-glycans, with core α 1,3 fucosyl residues that act as potential allergens. Any structural changes in these proteins may affect their bioactivity and/or bioavailability and may increase the side effects associated with their use [1,2,3,4] To avoid these shortcomings, despite their high production costs, most recombinant proteins used in humans are produced using mammalian cell lines. A cost-effective method of producing recombinant proteins for veterinary medicine involves the use of the Despite the cost advantages of the baculovirus expression system, there is concern with the structures of the N-glycans attached to the recombinant protein products. These are insect-type N-glycans, with a high percentage of core α 1,3 fucosyl residues. These core α 1,3 fucosyl residues have been found to induce allergic reactions

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