Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners.
Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.
Kim
Seonghun
S
School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, Republic of Korea.
Lee
Sun Bok
SB
D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility; D048229:Sulfolobus solfataricus / Q000201:enzymology; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D013879:Thioredoxins; D005982:Glutathione Transferase; D006836:Hydro-Lyases; C020576:gluconate dehydratase