Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions

Abstract

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.

FIG. 1

Cloning scheme of the bacterial expression vector pBM1.1. The expression module (a) is composed of the signal peptide of the pectate lyase gene (pelB), the IPTG-inducible T7 lac operon from the original pET vector (36), the synthetic His₁₀ cluster (His) (20), the variable-region genes (V_H and V_L) connected by (Gly₄Ser)₃ (linker), and the ETA′ gene. Plasmid pBM1.1 (b) contains the expression module, a kanamycin resistance gene (Kan^r), an E. coli origin of replication (pBR322 origin), an M13 origin of replication (f1 origin), and the lactose repressor gene (lacI). The binding single-chain variable fragments (scFv) are fused to ETA′.

FIG. 2

Purification of Ki-4(scFv)-ETA′. (a) 10% SDS-PAGE gel at different steps of purification. Lanes: M, molecular mass markers (with values in kilodaltons shown on the left); 1, soluble periplasmic content; 2, IMAC flowthrough; 3, IMAC eluate eluted with 250 mM imidazole; 4, Ki-4(scFv)-ETA′ after size exclusion chromatography. The gel was stained with Coomassie brilliant blue. (b) Corresponding Western blot. The recombinant immunotoxin was detected using the anti-ETA′ MAb TC-1 combined with alkaline-phosphatase-conjugated anti-mouse IgG MAbs and fast red. Arrowheads indicate the positions of Ki-4(scFv)-ETA′.

FIG. 3

Comparative analyses of polyclonal anti-GroEL immunoprecipitates. (a) 10% SDS-PAGE of precipitated proteins; (b and c) Western blot analyses of Ki-4(scFv)-ETA′ and GroEL, respectively. Results are shown for standard growth conditions (LB medium with 150 mM NaCl) (lanes 1 through 3) and osmotic-stress growth conditions in the presence of 10 mM glycine betaine (lanes 4 through 6). Lanes 1 and 4, before stress and IPTG induction; lane 2, 1/2 h before IPTG induction; lane 5, 1/2 h after supplementation with NaCl, sorbitol, and glycine betaine; lanes 3 and 6, 2 h after IPTG induction. Arrowhead indicates the position of Ki-4(scFv)-ETA′.

FIG. 4

Functional activity of purified recombinant immunotoxins after size exclusion chromatography using Bio-Prep SE-100/17 columns. (a) Elution profile monitored at 280 nm (solid line) combined with binding activity of the eluted fractions as documented by ELISA (dashed line). Immobilized rhCD30 receptor was incubated with dilutions of fractions from the column. Specifically bound Ki-4(scFv)-ETA′ was detected after incubation with the anti-ETA′ MAb TC-1 followed by alkaline-phosphatase-conjugated anti-mouse IgG. Converted substrate (o-phenylenediamine-dihydrochloride) was measured as absorbance at 405 nm. (b) Cell-binding activities of RFT5(scFv)- and Ki-4(scFv)-ETA′ as evaluated by flow cytometry analysis. (i) CD25⁺ CD30⁺ Hodgkin-derived L540Cy cells were incubated with PBS (open), and CD25⁻ CD30⁻ Hodgkin-derived HD-MyZ cells (shaded) or L540Cy cells (solid) were incubated with RFT5(scFv)-ETA′, for 15 min at 4°C. (ii) L540Cy cells were incubated with PBS (open), and HD-MyZ (shaded) or L540Cy (solid) were incubated with Ki-4(scFv)-ETA′, for 15 min at 4°C. Cells were stained with TC-1, mouse anti-TC-1, and goat anti-mouse FITC-conjugated antibody. Immunofluorescence (FL1 channel) was measured by flow cytometry, using a FACScan.

FIG. 5

Growth inhibition of Hodgkin-derived cell lines after incubation with recombinant immunotoxins as documented by cell viability assays. L540Cy (CD25⁺ CD30⁺) and HD-MyZ (CD25⁻ CD30⁻) cells were treated with the immunotoxins, and their abilities to metabolize the tetrazolium salt XTT to a water-soluble formazan salt (formed by mitochondrial dehydrogenase activity) were measured as absorbance at 450 and 650 nm (reference wavelength) in a cell viability assay (24). (a) Cytotoxic activities of 1:10 dilutions of RFT5(scFv)-ETA′ on L540Cy cells. (Inset) RFT5(scFv)-ETA′ (10 μg/ml in PBS) showed 100% binding activity on immobilized CD25 receptor; binding was reduced to <15% after the addition of 10 μg of soluble CD25 rCD25R receptor/ml. (b) Cytotoxic activity of Ki-4(scFv)-ETA′ on L540Cy cells (competition with MAb Ki-4).

FIG. 6

Cryoprotection and stabilization of 1 μg of CD30-binding proteins/ml by compatible solutes were measured as relative binding activity by a CD30 receptor ELISA. Specifically bound Ki-4(scFv)-ETA′ was detected after incubation with the anti-ETA′ MAb TC-1 followed by alkaline-phosphatase-conjugated anti-mouse IgG, and bound MAb Ki-4 was detected by alkaline-phosphatase-conjugated anti-mouse IgG. Converted phosphatase substrate (o-phenylenediamine-dihydrochloride) was measured as absorbance at 405 nm. (a) Relative binding activity of Ki-4(scFv)-ETA′ (open bars) compared to that of MAb Ki-4 (solid bars) after several rounds of freeze-thawing. (b) Relative binding activities of Ki-4(scFv)-ETA′ (open bars) and MAb Ki-4 (solid bars) in the presence of 1 M hydroxyectoine.