Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia co/i and its “oxidizing” mutant

AIM: To study the influence of redox environment of Escherichia coli ( E. coli) cytoplasm on disulfide bond formation of recombinant proteins.METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF,and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3)and M15[pREP4]respectively. At the same time, both plasmids were transformedinto a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.RESULTS: All recombinant E. colistrains could efficiently produce target proteins. The level of BbFGF in BL21(DE3)was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm,and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3)was higher than its counterpart from BL21(DE3). The ED50of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami[pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv]was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from indusion body in M15[pQE-H Fv] was30-35 mg/L and the affinity constant was 1.98×107 mol/L.There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.

Isospin effects of projectile fragmentation in a Boltzmann-Langevin approach

The isospin effects of projectile fragmentation at intermediate energies are investigated using an isospin-dependent Boltzmann-Langevin model.The collisions of mass-symmetric reactions including 58Fe,58Ni+58Fe,and Ni at intermediate energies,in the 30 to 100 MeV/A range,are studied for different symmetry energies.Yield ratios of the isotopic,isobaric,and isotonic pairs of fragments from the intermediate-mass region using three symmetry energies are extracted as functions of the N/Z ratio of the composite systems in the entrance channel and the incident energies.It is found that the yield ratios are sensitive to symmetry energies,especially for neutron-rich systems,and the calculations using soft symmetry energy are closer to the experimental data.The isospin effect is stronger for the soft symmetry energy,owing to the competition of the repulsive Coulomb force and the symmetry energy attractive force on the proton.For the first time,the splits are presented,revealing a transition from the isospin equilibrium at lower energies to translucency at intermediate energies.The results show a degree of transparency in that intermediate mass fragments undergo a transition from dependence on the composite systems in the entrance channel to reliance on the projectile and target nuclei.