title: An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100.
abstract: Targeting recombinant proteins at highly extracellular production in the culture medium of Escherichia coli presents a significant advantage over cytoplasmic or periplasmic expression. In this work, a recombinant protein between ZZ protein and alkaline phosphatase (rZZ-AP) was constructed. Because rZZ-AP has the IgG-binding capacity and enzymatic activity, it can serve as an immunoreagent in immunoassays. However, only a very small portion of rZZ-AP is generally secreted into the aqueous medium under conventional cultivation procedure. Hence, we emphasized on the optimization of the culture procedures and attempted to dramatically enhance the yield of extracellular rZZ-AP from E. coli HB101 host cells by adding sucrose, glycine, and Triton X-100 in the culture medium. Results showed that the extracellular production of rZZ-AP in the culture medium containing 5% sucrose, 1% glycine, and 1% Triton X-100 was 18.6 mg/l, which was 18.6-fold higher than that without the three chemicals. And the β-galactosidase activity test showed that the increased extracellular rZZ-AP was not due to cell lysis. Further analysis suggested a significant interaction effect among the three chemicals for the enhancement of extracellular production. Ultrastructural analysis indicated that the enhancement may be due to the influence of sucrose, glycine, and Triton X-100 on the periplasmic osmolality, permeability, or integrity of the cell wall, respectively. This proposed approach presents a simple strategy to enhance the extracellular secretion of recombinant proteins in the E. coli system at the process of cell cultivation.
authors.affiliation: School of Pharmacy, Weifang Medical University, 261053 Weifang, People's Republic of China.
authors.forename: Ru-Meng
authors.initials: RM
authors.lastname: Bao
authors.affiliation: School of Pharmacy, Weifang Medical University, 261053 Weifang, People's Republic of China.
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Yang
authors.affiliation: School of Pharmacy, Weifang Medical University, 261053 Weifang, People's Republic of China.
authors.forename: Chang-Mei
authors.initials: CM
authors.lastname: Yu
authors.affiliation: School of Pharmacy, Weifang Medical University, 261053 Weifang, People's Republic of China.
authors.forename: Wei-Fen
authors.initials: WF
authors.lastname: Zhang
authors.affiliation: School of Pharmacy, Weifang Medical University, 261053 Weifang, People's Republic of China. Electronic address: tangjb@wfmc.edu.cn.
authors.forename: Jin-Bao
authors.initials: JB
authors.lastname: Tang
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005998:Glycine / Q000494:pharmacology*; D017830:Octoxynol / Q000494:pharmacology*; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013395:Sucrose / Q000494:pharmacology*
chemical_list: D011993:Recombinant Fusion Proteins; D013395:Sucrose; D017830:Octoxynol; D000469:Alkaline Phosphatase; D005998:Glycine
keywords: Escherichia coli; Extracellular secretion; Glycine; Sucrose; Synergistic effect; Triton X-100; ZZ protein-alkaline phosphatase fusion protein