Expression of Active Recombinant Human Tissue-Type Plasminogen Activator by Using In Vivo Polyhydroxybutyrate Granule Display

Recombinant human tissue plasminogen activator (rPA) is a truncated version of tissue plasminogen activator (tPA), which contains nine disulfide bonds and is prone to forming inactive inclusion bodies when expressed in bacteria. To obtain functional rPA expression, we displayed the rPA on the surface of polyhydroxybutyrate (PHB) granules using phasin as the affinity tag. rPA was fused to the N terminus of the phasin protein with a thrombin cleavage site as the linker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis showed that rPA fusion was successfully displayed on the surface of PHB granules. An activity assay indicated that the rPA fusion is active. The in vivo surface display strategy for functional rPA expression in Escherichia coli is distinct for its efficient folding and easier purification and may be expanded to the expression of other eukaryotic proteins with complex conformation.Tissue plasminogen activator (tPA) derives from a fibrinolytic system of blood vessel endothelial cells, activates plasminogen to form plasmin, and is an effective drug for thrombolytic therapy. Native tPA is composed of 527 amino acid residues with five structural domains and 17 disulfide bonds (19). Recombinant human tissue plasminogen activator (rPA) is a variant version of tPA with nine disulfide bonds, consisting of kringle 2 and serine protease domain (12). rPA was confirmed to possess enhanced capability for thrombolysis compared with that of tPA. Therefore, rPA is more beneficial for the treatment of acute myocardial infarction (17, 26, 28).Heterologous expression of tPA as well as rPA in Escherichia coli often results in the formation of the insoluble aggregates known as inclusion bodies due to the multidisulfide bonds (3). The refolding of the inclusion bodies in vitro is a long and difficult task, especially for proteins with complex conformation and multiple disulfide bonds. In order to obtain directly the functional rPA from recombinant E. coli, many approaches have been utilized: expressing the rPA gene in E. coli trxB gor ahpC* mutant strains, of which the cytoplasm is highly oxidized; fusing the rPA gene with gpIII of ΦM13 and linking to the OmpA signal sequence, through which rPA is secreted into the medium; exploiting the novel twin-arginine translocation (Tat) pathway to obtain active rPA in the periplasmic space based on its inherent properties; and cosecreting of rPA with chaperones and adding low-molecular-size medium additives to promote the formation of disulfide bonds (6, 11, 15, 25). However, the successful expression of rPA in its soluble or active form gives rise to another task: separation and purification of soluble active rPA from large amounts of other proteins in cytoplasm or medium.Normal protein purification typically involves several chromatographic steps. Each step can be costly and time-consuming (4). The development of simple and reliable methods for protein purification, which can be applied to arbitrary products, is therefore an important goal in bioseparation technology developments. One method that was recently developed is the addition of an affinity tag sequence to the target protein gene (13). It was demonstrated that heterologous proteins can be displayed actively on the surface of biopolyester granules in E. coli by fusing to the polyhydroxyalkanoate (PHA) synthase (PhaC), which serves as an affinity tag of PHA granules (21). PHA granules are carbon inclusions produced intracellularly by bacteria for coping with changing, often oligotrophic environments (1). These inclusions are composed of a hydrophobic polyester core and hydrophilic phospholipid membrane with many embedded proteins (24). Besides PhaC, phasins (namely PhaPs) are the main proteins tightly attached to the surface of polyhydroxybutyrate (PHB) granules, which can stabilize and prevent coalescence of separate PHB granules (22). Due to the inherent properties, PhaP has been used as the affinity tag in vivo to display recombinant proteins on the surface of PHB granules (5).In this study, we fused the rPA gene to the N terminus of phaP. A thrombin cleavage site was introduced between them to release rPA from PHB granules. The fusion gene was then expressed in engineered E. coli, which was conferred with the PHB production pathway by cloning the PHB biosynthesis genes. We confirmed that recombinant rPA fusion was able to be actively expressed in vivo on the surface of PHB granules.