Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies

Effect of fertilization systems on soil microbial biomass and mineral nitrogen during canola (Brassica napus L.) development stages

The dynamics of microbial biomass and mineral nitrogen (N) during two growth stages were compared in soils under different fertilization treatments. At stem elongation, the fertilizer system had more N in the mineral pools, whereas manure and integrated systems had more N in the microbial biomass, indicating shifts in N pools between the two systems. At the flowering stage, integrated (manure + fertilizer) and manure systems had more N in two pools, indicating release of plant-available N from the microbial biomass. The carbon (C)/N ratio of the microbial biomass was significantly greater in manure and integrated treatments than in fertilizer treatments. Soil mineral N was significantly positively correlated with seed yield at the stem elongation stage, whereas it was positively correlated with soil microbial biomass C at the flowering stage. Stepwise regressions revealed that seed yield was significantly associated with mineral N at stem elongation and microbial biomass N at the flowering stage. © Taylor & Francis Group, LLC