Enhancing Functional Expression of Heterologous Burkholderia Lipase in Escherichia coli

Abstract

Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm of E. coli was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in enhancing the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm of E. coli formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

Bacteriophage T7 protein kinase: Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro

“…Based on the high amounts of T7PK that can be produced in E. coli from a single copy gene under control of a T7 promoter [19], T7PK has been used as a fusion tag to enhance the production of soluble recombinant proteins [23,24]. If combined with prior phosphatase treatment, autophosphorylation can provide a convenient route to the 32 P-labeling of T7PK-tagged proteins.…”

Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

“…The first one was to produce and purify both the lipase and its chaperone separately in E. coli and the fully active lipase was subsequently achieved by in vitro refolding process using various approaches [11,16-20]. Recently, the second strategy was directed to produce in vivo functional lipases in heterologous host E. coli using two-plasmid co-expression system without the need of in vitro refolding [21-24]. To date, expression of in vivo functional lipase in heterologous host E. coli using a dual expression cassette plasmid system has not been achieved.…”

Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus

“…Therefore, in vivo co-expression of LipBL with the chaperone GroEL/GroES, which assists in folding de novosynthesized proteins and disaggregating and refolding improperly folded proteins trapped in insoluble aggregates (Ewalt et al, 1997;Hartl & Hayer-Hartl, 2002), yielded catalytically active LipBL in E. coli BL21(DE3). Coexpression of GroEL has also been successfully applied to enhance the functional expression of other lipases (Narayanan et al, 2011;Shuo-Shuo et al, 2011) in E. coli. We have previously reported a laborious four-step protocol for chromatographic purification of recombinant LipBL Laane et al (1987).…”