Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.
The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.
Hayashi
Kokoro
K
Nara Institute of Science and Technology, Takayama, Ikoma, Japan.
Kojima
Chojiro
C
D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D013879:Thioredoxins / Q000378:metabolism
D058619:Maltose-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C011083:polyproline; D013879:Thioredoxins; D011392:Proline; D005982:Glutathione Transferase