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.
Geng
Yanping
Y
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
Wang
Shengjun
S
Qi
Qingsheng
Q
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D006885:Hydroxybutyrates; D037121:Plant Lectins; D011091:Polyesters; D000091402:Prohibitins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics
D006885:Hydroxybutyrates; C000717328:PHB protein, human; D037121:Plant Lectins; D011091:Polyesters; D000091402:Prohibitins; D011993:Recombinant Fusion Proteins; C008825:phasin; D010959:Tissue Plasminogen Activator