Abstract
BackgroundNeurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of hCNTF necessitate a recombinant expression system to produce large amounts of functional protein in soluble form. Earlier attempts to express hCNTF in Escherichia coli (E. coli) were limited by low amounts and the need to refold from inclusion bodies.ResultsIn this report, we describe a strategy to effectively identify constructs and conditions for soluble expression of hCNTF in E. coli. Small-scale expression screening with soluble fusion tags identified many conditions that yielded soluble expression. Codon optimized 6-His-hCNTF construct showed soluble expression in all the conditions tested. Large-scale culture of the 6-His-hCNTF construct yielded high (10 – 20 fold) soluble expression (8 – 9 fold) as compared to earlier published reports. Functional activity of recombinant 6-His-hCNTF produced was confirmed by its binding to hCNTF receptor (hCNTFRα) with an EC50 = 36 nM.ConclusionOur results highlight the combination of codon optimization and screening soluble fusion tags as a successful strategy for high yielding soluble expression of hCNTF in E. coli. Codon optimization of the hCNTF sequence seems to be sufficient for soluble expression of hCNTF. The combined approach of codon optimization and soluble fusion tag screen can be an effective strategy for soluble expression of pharmaceutical proteins in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0092-x) contains supplementary material, which is available to authorized users.
Highlights
Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system
McDonald and co-workers have reported the presence of only 13% of soluble Human ciliary neurotrophic factor (hCNTF) in E. coli (BL21) cell extracts [23]
Soluble expression of the 6-His-(fusion tag)-hCNTFs obtained were (6-His)-hCNTF in all conditions tested points to the fact that codon optimization is sufficient for soluble expression of hCNTF in E. coli
Summary
Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. The advantages are offset by the challenges poised by recombinant production of eukaryotic proteins [27,28] Bacterial hosts such as E. coli are often limited in amounts of tRNA for the codons that are used rather less frequently. Instances where codon bias of the gene is significantly different from that of the expression host E. coli, paucity of the tRNAs severely limits the translation process. This results in non-optimal translation of the RNA including stalling, termination, possible frame-shifting and low levels of protein expression [29,30]. Production of soluble proteins in E. coli remains largely a trial-and-error process
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