Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones

Abstract

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Fig 1.

Affinity purification and protease sensitivity to estimate native folding after salt + osmolyte treatment. Cells expressing plasmid-encoded E8R-NusA fusion protein were grown in the presence of IPTG at 20°C for 20 hours or at 37°C for 4 hours, as indicated. Salt treatment: 30 minutes before the IPTG induction, 0.5 M NaCl, and 5 mM betaine were supplemented to the medium. After cell lysis, homogenates were centrifuged. The insoluble pellets (left panel), supernatants (central left panel), or the ion metal affinity–purified fractions (central right panel) were separated on SDS-PAGE and stained by Coomassie blue. Right panel: total homogenates separated by SDS-PAGE and immunoblotted with DnaK antibodies. Lower panel: supernatant fractions (100 μg) were incubated with trypsin (5 μg) for 2 hours at 24°C before separation by SDS-PAGE and Coomassie blue staining. SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Fig 2.

Osmolytes or BA treatments but not heat shock improve IMAC yields of O36. Thirty minutes before induction, 0.5 M NaCl, 5 mM betaine, or K-glutamate (or all) were added as indicated. Cells were harvested 2 hours after induction and extracts were centrifuged, IMAC purified, separated on SDS-PAGE, and stained by Coomassie blue as in Figure 1. Total homogenates were separated by SDS-PAGE and immunoblotted with DnaK antibodies. Relative amounts of DnaK and O36 were estimated with a densitometer and the values reported in the table. A heat shock effect was induced by raising the growth temperature to 42°C for 30 minutes before IPTG induction. BA (10 mM) was added to a culture grown at 20°C, 20 minutes before IPTG induction (0.1 mM). Bacteria were recovered after 20 hours with BA at 20°C. Experiments were repeated at least thrice. BA, benzyl alcohol; IMAC, immobilized metal ion affinity chromatography; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Fig 3.

Complementary effects of GroES-GroEL, betaine, and BA on the solubility of the O15 protein. Cells expressing recombinant proteins O15, without or in the presence of a second compatible plasmid coexpressing the GroES-GroEL operon, were treated at 20°C for 20 hours in the presence of salt + betaine or of BA, as in Figure 2. Betaine and BA were added to the bacterial culture 30 and 20 minutes before IPTG induction, respectively. Soluble O15 protein was IMAC purified and detected as in Figures 1 and 2. Values are from 1 representative experiment out of 3. BA, benzyl alcohol; IMAC, immobilized metal ion affinity chromatography

Fig 4.

IMAC yields in the presence of different chaperones and osmolytes. E8R was expressed as in Figure 1 in the absence or presence of compatible plasmids coexpressing DnaK-DnaJ-GrpE and ClpB (KJEB), GroES-GroEL (LS), and IbpA/B (IAB). NaCl and osmolytes were added 30 minutes before IPTG induction (0.1 mM) and the cells were grown 20 hours at 20°C. Then, IPTG was removed by centrifugation from the culture medium and the cells were grown for 2 additional hours at 20°C to allow chaperone-mediated disaggregation. The E8R protein was purified by IMAC and detected as in Figures 1–3. Values are from 1 representative experiment out of 3. IMAC, immobilized metal ion affinity chromatography

Fig 5.

Improved IMAC yields using molecular chaperones and BA. In a preliminary screening to identify optimal conditions for expressing soluble target proteins, cells harboring each of the 6 target proteins, were cotransformed with different combinations of chaperone plasmids (I: KJEB + LS, II: KJEB + LS + IAB), as in Figure 4. BA treatment, protein IMAC purification and detection was as in Figures 1–4. Values are from 1 representative experiment out of 3. BA, benzyl alcohol; IMAC, immobilized metal ion affinity chromatography