Lectin-Based Affinity Tag for One-Step Protein Purification

Abstract

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.

Glycosylation Is Required for Outer Membrane Localization of the Lectin LecB inPseudomonas aeruginosa

“…We thus named this variant high-molecular-weight LecB (HW LecB) to distinguish it from its low-molecular-weight variant (LW LecB) and asked whether this HW LecB variant was also functional in binding to mannose. This binding capability of HW LecB was assayed by purification of both variants from the periplasm by the same affinity chromatography which we established earlier (28,51). Both variants were present in the purified lectin fraction in comparable amounts, showing that HW LecB also binds quantitatively to mannose and can be copurified with the low-molecular-weight variant from the periplasm (Fig.…”

“…Functional LecB from the periplasm was purified from 500 mg of cells (dry weight) by its affinity toward mannose as described previously (51). Mannose was removed by repeated ultrafiltration (Vivaspin 6, 5-kDa cutoff; Vivascience, Hannover, Germany) and washed with 100 mM Tris-HCl (pH 8.0).…”

Lectin-mediated reversible immobilization of human cells into a glycosylated macroporous protein hydrogel as a cell culture matrix

“…oligosaccharides or functionalized dendrimers 56, 57 ) can be used to disperse those structures and release the cells. YFP-LecB can be efficiently produced in E. coli 34 und purified via a D-mannose affinity chromatography, which makes the production easy and permits the unlimited production of the adaptor molecule. One major advantage of the affinity purification is the in-process control of the proteins functionality: the binding of YFP-LecB to the column indicates the correct folding and functionality of the tetrameric lectin B domain, while its fluorescence directly correlates with the functionality of the YFP reporter domain.…”

“…For each experiment the expression strain was freshly transformed with 1 µl YFP-LecB plasmid 34 (180 ng/µl) being mixed with chemical competent E. coli BL21 Tuner (Merck Millipore, Darmstadt, Germany), incubated 45 min on ice followed by 42 °C for 1 min and subsequent incubation on ice for 3 min 800 µl of pre-warmed lysogeny broth (LB) medium was added and cells were incubated at 37 °C for 1 h, followed by plating 50 µl of cell suspension on selective LB-amp (100 µg/ml) medium and incubation over night at 37 °C. The next day, one clone was picked and transferred to 50 ml selective LB-amp medium and grown at 37 °C overnight.…”

“…Tielker et al . have recombinantly produced LecB in Escherichia coli and established a purification method through mannose agarose beads to explore the potential of LecB as a tag for single-step protein purification 34 . Furthermore, they constructed a YFP-LecB fusion protein containing LecB as a binding domain and YFP as a reporter for different possible applications.…”