JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 20:677–683 (2000) Mary Ann Liebert, Inc. Antiviral and Antiluteolytic Activity of Recombinant Bovine IFN-v1 Obtained from Pichia pastoris O. BOUÉ, 1 J.L. GARCÍA, 1 M. VILLAR, 1 K. ALAZO, 1 A. PÉREZ, 1 E. RAMOS, 1 C. MORALES, 2 O.L. MORERA, 2 M. REDONDO, 3 C. MONTERO, 3 and M. RODRÍGUEZ 3 ABSTRACT The gene coding for bovine interferon-v 1 (BoIFN- v 1) was recently cloned and expressed at high levels in the yeast Pichia pastoris . The recombinant BoIFN- v1 protein shows antiviral activity in different cell lines and has an antiluteolytic effect in cyclic ewes. In this article, we describe a method for purification of BoIFN- v 1 expressed in the methylotrophic yeast P. pastoris and characterization of its activity in vivo. The recombinant protein secreted to the culture medium had low activity because of self-aggregation. BoIFN- v1 was solubi- lized using urea and desalting and finally purified by ion exchange chromatography on Q-Sepharose Fast Flow. The yield of purified product was approximately 300 mg/L of fermentation culture, with a specific an- tiviral activity of 10 8 IU/mg. Its purity was at least 80%. The biologic characterization of purified BoIFN- v1 was determined by induction of an antiviral state on ewes challenged with 100 lethal doses (LD) of Aujeszky virus and by the extension of the corpus luteum life span and interestrous interval in cyclic cows. Ewes treated with 2 3 10 6 IU/kg BoIFN- v 1 were protected from Aujeszky virus infection. In cows receiving an intrauter- ine infusion of 1 mg BoIFN- v 1, equally distributed between the two uterine horns, twice daily from day 14 to day 22 of the experimental estrous cycle, the lifespan of the corpus luteum (25 vs. 19 days) and the inter- estrous intervals (26 vs. 21 days) were extended when compared with a control group (p , 0.05). We show that recombinant BoIFN- v 1 purified from P. pastoris has high antiviral activity and is an effective antilute- olytic agent in cattle. 677 INTRODUCTION S UBOPTIMAL REPRODUCTIVE PERFORMANCE in livestock ac- counts for millions of dollars in losses to producers yearly. (1–4) Most of the embryonic wastage occurs during early pregnancy, which is the most critical stage for survival of the conceptus. Roughly two thirds of the total prenatal deaths in domestic animals occur before day 30 of gestation. (5–7) Mea- sures aimed at reducing embryo mortality in ruminants require an understanding of the control mechanisms of growth, differ- entiation, and implantation of the conceptus. Estrous cyclicity in domestic ruminants is dependent on uter- ine endometrial secretion of prostaglandin F 2a (PGF 2a ), which is transported to the ovaries by a local venoarterial pathway, where it causes regression of the corpus luteum (CL). Proges- terone production cessation resulting from luteal regression re- moves the progesterone block to ovulation and permits initia- tion of a new cycle. In most eutherian mammals, the CL life span must be extended during early pregnancy so that proges- terone production is maintained. (8–11) The steroid hormone acts on the mucosal lining of the uterus to provide an environment in which the conceptus can continue to develop. The presence of a viable conceptus in the uterus alters maternal endocrinol- ogy, resulting in luteal maintenance and other metabolic and immunologic changes. The functional life span of CL in rumi- nants is extended by pregnancy recognition signals from the trophoblast, which act in a paracrine manner to abrogate the mechanism responsible for the pulsatile release of the PGF re- quired for luteolysis (antiluteolytic signal). (12–14) This tro- phoblast signal is mediated by a novel family of type I inter- ferons (IFN), IFN-t. The unique function of this protein in maintaining pregnancy in ruminants has been the subject of sev- eral reviews. (9–11,15,16) The expression of IFN-t is regulated developmentally to the period of pregnancy recognition, that is, days 15–24 in cows. (15–17) It has been implicated in triggering a series of ma- Division of 1 Technological Development and 3 Mammalian Cell Genetics, Center for Genetic Engineering and Biotechnology, 10600 Havana, Cuba. 2 Centro Nacional de Sanidad Agropecuaria, San José de las Lajas 32700, Havana, Cuba.