Lectin-based affinity tag for one-step protein purification.
The production of pure protein is indispensable for many applications in life sciences, however protein purification protocols are difficult to establish, and the experimental procedures are usually tedious and time-consuming. Therefore, a number of tags were developed to which proteins of interest can be fused and subsequently purified by affinity chromatography. We report here on a novel lectin-based affinity tag using the D-mannose-specific lectin LecB from Pseudomonas aeruginosa. A fusion protein was constructed consisting of yellow fluorescent protein and LecB separated by an enterokinase cleavage site. This protein was overexpressed in Escherichia coli Tuner (DE3), and the cell extract was loaded onto a column containing a mannose agarose matrix. Electrophoretically pure fusion protein at a yield of 24 mg/L culture was eluted with a D-mannose containing buffer The determination of equilibrium adsorption isotherms revealed an association constant of the lectin to the mannose agarose matrix of Ka = 3.26 x 10(5)/M. Enterokinase treatment of the purified fusion protein resulted in the complete removal of the LecB-tag. In conclusion, our results indicate that the lectin LecB of P. aeruginosa can be used as a tag for the high-yield one-step purification of recombinant proteins.
Tielker
Denis
D
Heinrich-Heine-University Duesseldorf, Juelich, Germany.
Rosenau
Frank
F
Bartels
Kai-Malte
KM
Rosenbaum
Thomas
T
Jaeger
Karl-Erich
KE
D000327:Adsorption; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005821:Genetic Techniques; D007700:Kinetics; D037102:Lectins / Q000737:chemistry*; D008164:Luminescent Proteins / Q000378:metabolism; D008358:Mannose / Q000737:chemistry; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D001426:Bacterial Proteins; D037102:Lectins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D004765:Enteropeptidase; D008358:Mannose