title: Supplementation of substrate uptake gene enhances the expression of rhIFN-β in high cell density fed-batch cultures of Escherichia coli.
abstract: Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the "ara" promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-β (rhIFN-β) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-β in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK (+)) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-β in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-β was obtained with the modified strain which was 35 % higher than the control.
authors.affiliation: School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. anuradha.bharara@gmail.com
authors.forename: Anuradha B
authors.initials: AB
authors.lastname: Singh
authors.forename: Krishna J
authors.initials: KJ
authors.lastname: Mukherjee
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods; D002452:Cell Count; D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005991:Glycerol Kinase / Q000235:genetics / Q000378:metabolism; D016899:Interferon-beta / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D003470:Culture Media; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D016899:Interferon-beta; D005991:Glycerol Kinase