Production and purification of recombinant buffalo leukemia inhibitory factor (rBuLIF) from stably transfected COS-1 cells

Abstract

Leukemia inhibitory factor (LIF) is a secreted glycoprotein that was initially identified through its ability to induce macrophage differentiation in the murine M1 myeloid leukemic cell line. LIF also maintain pluripotency in Embryonic Stem cells (ESCs). Mouse LIF (mLIF) is in use for buffalo stem cells production whose efficiency is unclaimed. We successfully cloned the buffalo LIF gene in pAcGFP-N1 vector which was subsequently transfected into eukaryotic cell line (COS-1). After several rounds of selection in the presence of G418 and single cell clonal expansion, a stably transfected cell line of COS-1_BuLIF was developed which expressed BuLIF_GFP at high level. The strong expression of BuLIF was observed at 70th passage and cells could grow in the absence of selection pressure without losing GFP signal. PCR based identification to detect GFP and BuLIF gene in the COS-1_BuLIF genome further confirmed the genomic integration of BuLIF. Co-immunoprecipitation technique was employed for the purification of rBuLIF_GFP where Cynogen Bromide activated Sepharose was used as matrix to couple with anti GFP monoclonal antibody. Pure BuLIF_GFP protein was obtained from several T75 cm2 confluent flasks. The SDS-PAGE revealed a single band of around 65-70kDa molecular weight protein which corresponds to the expectedmolecular weight of BuLIF_GFP fusion protein (22.9kDa+35.0 kDa=55.9kDa). LIF protein is highly glycosylated which increase the size of protein from 10-30kDa. The purified protein will further confirmed by Mass spectrometry based identification (Q-TOF) which revealed the identity of protein as BuLIF. Thus, this work demonstrates the successful purification of rBuLIF_GFP which may find its applications in bovine stem cell and related biological field.