RESUMO
Background: Pivotal roles of Nerve growth factor [NGF] in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human beta-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use of E. coli as an expression host for a protein with three intra-chain disulfide bonds were evident
Objectives: Here an optimized system was developed to overexpress the soluble NGF in E. coli
Materials and Methods: The gene encoding the beta subunit of mature hNGF was optimized based on E. coli codon preference and cloned into pET-32a expression vector providing His- and Trx- tags for detection and increasing the solubility of recombinant protein, respectively. The recombinant DNA was expressed in E. coli Origami [DE3], which enhances the correct formation of disulfide bonds in the cytoplasm of E. coli. Different culture conditions were evaluated to increase soluble expression of the target protein
Results: The highest soluble expression level was achieved when E. coli Origami [DE3] cells expressing NGF were grown at 30[degree]C in TB medium with 0.2 mM IPTG induction at OD[600nm] = 1 for 4 h
Conclusions: Our results indicated that the recombinant NGF was successfully expressed as a soluble form