Establishment of a Large-Scale Purification Procedure for Purified Recombinant Bovine Interferon-.TAU. Produced by a Silkworm-Baculovirus Gene Expression System

Abstract

ABSTRACT. We developed a procedure for the large-scale purification of bovine interferon-τ (boIFN-τ) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-τ (rboIFN-τ) was efficiently produced in the silkworm infected with boIFN-τ cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 × 10 8 U/mg) of 91% pure rboIFN-τ by means of a combination of the ANT, followed by QSC and CSCC. KEY WORDS: baculovirus, bovine, IFN-τ, purification, silkworm.J. Vet. Med. Sci. 66(11): 1395-1401, 2004 Interferon (IFN)-τ, originally named trophoblast protein-I, is the major secretory protein of trophoblastic tissues and plays an important role in the maternal-fetal recognition of pregnancy in ruminants [1,8,19]. Interferon-τ has been classified as a member of the large multi-gene type I IFN family, and has about 85 and 70% overall homology to IFN-ω on the bases in the cDNA nucleotide and amino acid sequences, respectively [4,9,20]. As expected from the homology, IFN-τ possesses most of the major functions of IFNs, including anti-viral activity, but does not lead to a febriferous reaction, a serious side effect of the IFN therapy. Because of its biological activities, IFN-τ is applied as a useful tool in ongoing studies on the establishment of pregnancy, and is also expected to be used as an agent to improve the pregnancy rate through the control of implantation and to treat viral diseases. For these applications, it is essential to establish a mass production procedure for purified IFN-τ. Up to now, recombinant ovine and bovine IFN-τ boIFN-τ) had been expressed by baculovirus, yeast and Escherichia coli (E.coli) gene expression systems [3,6,16]. Among these, the baculovirus gene expression system is the most suitable system for producing recombinant IFN-τ for animal experiments and practical biological agents because of its similarity in terms of post-translational modifications, such as glycosylation, phosphorylation, conformation, antigenicity and so on [14]. As the baculovirus gene expression system, Autographa californica (Ac) nuclear polyhedrosis virus (NPV) with insect culture cells as the host and Bombyx mori (Bm) NPV with silkworm larvae as the host were popularly used. The advantage of the AcNPV-insect cell culture system is the absence of serum protein contamination in the culture fluids, since the cells can be cultured wit...

Production of a recombinant antibody fragment in whole insect larvae

“…While antibodies have been produced in baculovirus expression systems [3,8,15,16,26,30], and a number of other proteins have been expressed in insect larvae, including IL-2 [22], interferon-tau [20], cardiac sodium-calcium exchange protein [7], GFP [4], chymase [29], and butyrylcholinesterase [24], we believe this is the first example of the expression of a recombinant antibody in whole insect larvae.…”

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae

“…However, neither of these systems is suitable for mass production of recombinant proteins. Some methods were established for purification of recombinant proteins from the hemolymph (23,(26)(27)(28)(29). We have purified >90% pure rhGM-CSF from the hemolymph with a combined method of salt precipitation, gel filtration, and Ni-chelating Sepharose chromatography (unpublished data).…”

Interferons: application in veterinary medicine

“…Він показав високу противірусну актив-ність як у культурах клітин ВРХ та овець, так і в котячих та людських культурах клітин [95], причому його токсична дія була менш вираже-ною порівняно з дією людського інтерферону альфа [97]. Були розроблені методи очистки та широкомасштабного виробництва цього цито-кіну [57,82], і його почали використовувати для боротьби з інфекційними хворобами тварин, передусім ВРХ та овець [58].…”