title: Efficient extracellular production of κ-carrageenase in Escherichia coli: effects of wild-type signal sequence and process conditions on extracellular secretion.
abstract: Signal peptides direct proteins to translocate across the bacterial cytoplasmic membrane. This study aimed to improve the level of extracellular secretion of recombinant carrageenase by recombining the gene encoding wild-type signal peptide (OmpZ) of Zobellia sp. ZM-2 κ-carrageenase into the expression vector pProEX-HTa-cgkZ. The recombinant strain BL21-HTa-cgkZ achieved extracellular secretion of κ-carrageenase. The effects of induction, culture conditions, and additives were investigated to further promote the extracellular secretion of the enzyme. Results showed that the wild-type signal sequence secreted recombinant κ-carrageenase out of the cytoplasmic membrane. Low temperature (23 °C) and optimum isopropyl-β-thiogalactoside concentration (0.9 mM) favored soluble protein expression. Moreover, additives such as lactose, glycine, Tween-80, and TritonX-100 promoted the release of intracellular enzymes. The existence of OmpZ resulted in 51% of the total κ-carrageenase accumulation secreted into culture medium, and 33% accumulated in the periplasmic space. High extracellular secretion of recombinant κ-carrageenase under the optimum conditions showed promising applications of the process for extracellular protein production.
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Tian
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China. Electronic address: mousun@ouc.edu.cn.
authors.forename: Haijin
authors.initials: H
authors.lastname: Mou
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001483:Base Sequence; D002351:Carrageenan / Q000096:biosynthesis; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside; D008969:Molecular Sequence Data; D017830:Octoxynol; D011136:Polysorbates; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D055571:Secretory Pathway / Q000502:physiology*; D013696:Temperature
chemical_list: D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D011136:Polysorbates; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D002351:Carrageenan; D017830:Octoxynol; D006026:Glycoside Hydrolases
keywords: Escherichia coli; Extracellular secretion; OmpZ; pProEX-HTa-cgkZ; κ-Carrageenase