title: Novel strategy for production of aggregation-prone proteins and lytic enzymes in Escherichia coli based on an anchored periplasmic expression system.
abstract: For over 2 decades, Escherichia coli has been successfully used for the production of various recombinant proteins. However, several technical limitations have influenced the extent of recombinant protein expression in the E. coli host because of (i) heterologous protein accumulation often observed in inactive inclusion bodies either in the cytoplasm or periplasm, or (ii) lytic activity of recombinant proteins, which causes cell lysis, that hinder high production yield. We developed a novel strategy for the efficient production of aggregation-prone proteins and lytic enzymes in the E. coli host. For this purpose, we used an anchored periplasmic expression (APEx) system, in which target proteins are produced in the periplasm and tethered on the inner membrane. Protein aggregation and lytic activity can be prevented through anchoring of individual proteins to the inner membrane. Two model proteins (aggregation-prone human leptin and lytic Pseudomonas fluorescens SIK W1 lipase) were examined, and both proteins were successfully produced and anchored to the inner membrane under optimized culture conditions. Upon expression, the inner membrane-anchored proteins were subjected to simple purification procedures; the proteins were confirmed to be of high purity and bioactivity.
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Jae Hyung
authors.initials: JH
authors.lastname: Lee
authors.forename: Natarajan
authors.initials: N
authors.lastname: Velmurugan
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
mesh_terms: D055162:Biocatalysis; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics*; D006801:Humans; D055767:Immobilized Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism; D020738:Leptin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D050296:Microbial Viability; D019897:Periplasm / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D055767:Immobilized Proteins; D020738:Leptin; D011994:Recombinant Proteins; D008049:Lipase
keywords: Anchored periplasmic expression; Escherichia coli; Leptin; Lipase; Periplasm