Codon optimization and factorial screening for enhanced soluble expression of human ciliary neurotrophic factor in Escherichia coli.
BACKGROUND
Neurotrophic 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.


RESULTS
In 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.


CONCLUSIONS
Our 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.
Jaakko M
JM
Itkonen
Arto
A
Urtti
Louise E
LE
Bird
Sanjay
S
Sarkhel
D020934:Ciliary Neurotrophic Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D012995:Solubility
D020934:Ciliary Neurotrophic Factor; D003062:Codon