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Publication Abstract from PubMed

In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 A resolution and also that of the [LSL(150):(lactose)beta, gamma)] binary complex at 1.67 A resolution. This complex reveals two lactose molecules bound to the beta and gamma sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150) (LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.

High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile beta-trefoil lectin domain from the mushroom Laetiporus sulphureus.,Angulo I, Acebron I, de las Rivas B, Munoz R, Rodriguez-Crespo I, Menendez M, Garcia P, Tateno H, Goldstein IJ, Perez-Agote B, Mancheno JM Glycobiology. 2011 Oct;21(10):1349-61. Epub 2011 Jun 1. PMID:21632870^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.