Production and Post-Translational Modification of Tissue Plasminogen Activator (tPA) in Namalwa Cells

N-linked glycosylation is a major post-translational event having profound influence on the quality of recombinant proteins destined for therapy and hence it is extensively studied [1]. Glycosylation is affected by the type of host cell, environmental stimuli, and protein structure [2]. This study will investigate the first two factors by characterising a model protein: tissue plasminogen activator (tPA) expressed in Chinese hamster ovary (CHO) and a human lymphoblastoid cell line (Namalwa). tPA is a fibrin-specific serine protease of plasminogen involved in the dissolution of vascular fibrin clots in vivo. It exists as a mixture of single-chain and two-chain (A and B) glycoproteins (66-72kDa) with four potential N-linked glycosylation sites at Asn117 (invariably high mannose type) Asn184 (variably occupied complex type) Asn218 (never glycosylated) and Asn448 (invariably complex type) and an O-linked fucose occurs at Thr61. Type I tPA has three sites occupied with N-linked oligosaccharides and type II has only two sites occupied [3].