Parameters influencing the expression of mature glial-cell-line-derived neurotrophic factor in Escherichia coli.
Factors governing expression in Escherichia coli, namely promoters, fusion partners, targeting signals, host strains, growth temperature of cultures and inducer concentrations, were investigated to elucidate their influence on the accumulation of mature glial-cell line-derived neurotrophic factor (GDNF). The present study provided evidence indicating that translational and/or post-translational events were more important in determining overall accumulation of the target protein than was transcription. Under the control of the strong inducible tac or T7 promoter, no direct correlation between transcript abundance and final yield of recombinant protein was observed. GDNF was also recalcitrant to being produced in a soluble form in E. coli. Direct expression resulted in the exclusive localization of GDNF in inclusion bodies, regardless of whether the protein was produced in the cytoplasm or targeted to the periplasm. The fusion approach was found to be the most efficient method, as it resulted not only in the highest level of GDNF produced, albeit primarily in inclusion bodies, but also in the accumulation of small amounts of soluble proteins. Using different host strains, low inducer concentration or sub-optimal growth temperature did not result in any detectable shift towards solubility. Persistent localization in inclusion bodies, low levels of expression as a native protein and in vivo proteolysis of soluble fusion forms appeared to be influenced by structural features located at the N-terminus of GDNF. Deletion of this region was found to result in substantial alleviation of these problems.
Liew
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Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore.
Choo
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003593:Cytoplasm; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D013696:Temperature; D014158:Transcription, Genetic / Q000235:genetics
D004269:DNA, Bacterial; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D015514:Oncogene Proteins, Fusion; D012329:RNA, Bacterial; D011994:Recombinant Proteins