Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli

Abstract

Periplasmic secretion has been used in attempts to construct an efficient whole-cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin-arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole-cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole-cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL-GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole-cell OPH activity of the chaperone-coexpressing cells was ∼5.5-fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat-based periplasmic translocation of OPH in the chaperone-coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole-cell activity of periplasmic OPH-secreting cells, yielding an effective whole-cell biocatalyst system with highly reduced diffusion limitations.

Secretory expression of organophosphorus hydrolase OPHC2 inYarrowia lipolyticaPolg

“…[18] In recent years, several reports have presented the successful translocation of OPH into the periplasmic space using the general secretory (Sec) pathway with the pelB signal sequence [19] and also using the twin-arginine translocation (Tat) pathway with the Tat signal sequence. [20][21][22] Moreover, recombinant OPH harboring a native Tat signal sequence can be secreted into the supernatant without a C-terminal tag. [23] However, lipopolysaccharide (LPS), a well-known The percentage data of protein purity were obtained using Quantity One Ò 1-D Analysis Software from Bio-Rad (Hercules, CA, USA).…”

A Robust Fractionation Method for Protein Subcellular Localization Studies in Escherichia Coli

“…compared periplasmic fractions obtained by EDTA/lysozyme and cold osmotic shock and demonstrated cross-contamination in each case [17,24]. Many publications show no due-diligence to fraction purity [18,19,21,25,26]. Some do utilize controls in the form of subcompartment specific host proteins as purity markers but are incomplete [27,28].…”

“…The periplasmic extraction is the most critical step of the process since disruption of the IM results in contamination from cytoplasmic proteins. Common methods use either an EDTA-lysozyme strategy [17,18] or cold osmotic shock [19,20], or a combination of both [21]. In the former method, EDTA is used to destabilize the OM [22], allowing the lysozyme to enter the periplasm and hydrolyze the peptidoglycan cell wall.…”

“…Alkaline phosphatase (PhoA) variants were used as reporter proteins to demonstrate either cytoplasmic or periplasmic cellular localization. The former lacks a signal peptide whereas in the latter, PhoA was fused to either a Sec-specific [28,29] or Tat-specific signal peptide [19,21,26,28,30] for export. PhoA is naturally exported by the Sec pathway [28], but has also been shown to export into the periplasm as a fully folded protein via the Tat pathway when fused to a TorA signal peptide [26,28].…”

“…This process subsequently releases the periplasm and leaves the cells as spheroplasts [18]. The second method generates an osmotic shock induced by successively adding a hypertonic solution to weaken the OM, followed by a hypotonic solution to partially disrupt the OM without compromising the integrity of the IM [19]. …”