title: Stable expression and secretion of polyhydroxybutyrate depolymerase of Paucimonas lemoignei in Escherichia coli.
abstract: An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium. However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells. INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22 degrees C but was efficiently secreted into the extracellular medium when cultivated at 37 degrees C.
authors.affiliation: Department of Microbiology, Chungnam National University, Daejeon, 305-764, Republic of Korea.
authors.forename: Se Whan
authors.initials: SW
authors.lastname: Park
authors.forename: Moon Gyu
authors.initials: MG
authors.lastname: Chung
authors.forename: Hwa Young
authors.initials: HY
authors.lastname: Lee
authors.forename: Jeong Yoon
authors.initials: JY
authors.lastname: Kim
authors.forename: Young Ha
authors.initials: YH
authors.lastname: Rhee
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D042521:Burkholderiaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010957:Plasmids; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003470:Culture Media; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase