Express SUMO-B-(16)₄-Ferritin

Objective

To evaluate the expression and solubility of the SUMO-B-(16)₄-Ferritin composite protein in E. coli, and to determine whether the SUMO tag enhances the soluble yield of this nanoparticle immunogen.

Materials and Methods

1. Plasmid Construction

   The gene encoding SUMO-B-(16)₄-Ferritin was synthesized and cloned into the pET28a-sumo vector under the control of a T7 promoter. The construct includes an N-terminal 6×His tag for purification.

2. Host Strain

   E. coli BL21(DE3) cells were transformed with the recombinant plasmid.

3. Expression Conditions
   • Single colonies were inoculated into LB medium containing kanamycin and grown overnight at 37°C.
   • The overnight culture was diluted 1:100 into fresh LB + kanamycin and grown at 37°C until OD600 reached 0.6–0.8.
   • Protein expression was induced with IPTG at final concentrations of 0, 0.2, and 0.5 mM.
   • Cultures were incubated at 18°C and 37°C for 16 hours and 4 hours, respectively, to compare temperature effects.
4. Sample Preparation and Analysis
   • Cells were harvested by centrifugation and lysed by sonication.
   • Lysates were separated into supernatant (soluble fraction) and pellet (insoluble fraction) by centrifugation.
   • Protein samples from both fractions were analyzed by SDS-PAGE.

Results

SDS-PAGE Analysis

• Three recombinant ferritin nanoparticle immunogens were tested: B-(16)₄-Ferritin (this part), H1-(16)₄-Ferritin, and H3-H1-B-Ferritin.
• SDS-PAGE showed that for the SUMO-B-(16)₄-Ferritin construct, the target protein band appeared at the expected molecular weight in the total cell lysate.
• No significant soluble expression was detected in the supernatant for any of the tested IPTG concentrations (0, 0.2, 0.5 mM) or induction temperatures; the majority of the target protein was found in the insoluble pellet fraction.
• This result was consistent for all three constructs under the tested conditions.

Conclusion

• The SUMO tag did not significantly improve the soluble expression of the B-(16)₄-Ferritin fusion protein in E. coli under the tested conditions.
• Most of the expressed protein formed inclusion bodies, requiring further processing.

Troubleshooting and Further Steps

• To obtain functional protein, in vitro refolding was performed on the insoluble material to recover properly folded nanoparticles. (See detailed folding protocol and results here.)
• Potential improvements could include optimizing induction temperature, IPTG concentration, or testing other solubility-enhancing tags.

SDS-PAGE Analysis of Soluble Expression of Recombinant Ferritin-Based Immunogens

SDS-PAGE analysis of soluble protein expression for three recombinant ferritin nanoparticle immunogens (B-(16)₄-F, H1-(16)₄-F, and H3-H1-B-F) in E. coli under different IPTG induction concentrations. Lanes 1–3: B-(16)₄-F with 0, 0.2, and 0.5 mM IPTG; lanes 4–6: H1-(16)₄-F with 0, 0.2, and 0.5 mM IPTG; lanes 7–10: H3-H1-B-F with 0, 0.2, 0.5, and 1 mM IPTG. The molecular weight markers (Marker) are shown on the left. No significant soluble expression of the target proteins was detected in the supernatant under the tested induction conditions.

Western blot detection of in vitro folding product using anti His-tag primary antibody.

Summary

The SUMO-B-(16)₄-Ferritin composite part can be expressed in E. coli, but most of the product is insoluble under standard conditions, even with the SUMO tag. This highlights the challenge of expressing certain antigenic peptides and the need for subsequent protein refolding to obtain functional nanoparticles.