In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na(+)-citrate symporter CitS as an example.

@article{citeulike:1379682, abstract = {The determination of 3D structures of membrane proteins is still extremely difficult. The co-crystallization with specific binding proteins may be an important aid in this process, as these proteins provide rigid, hydrophilic surfaces for stable protein-protein contacts. Also, the conformational homogeneity of the membrane protein may be increased to obtain crystals suitable for high resolution structures. Here, we describe the efficient generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) as specific binding molecules for membrane proteins. We used both phage display and ribosome display to select DARPins in vitro that are specific for the detergent-solubilized Na(+)-citrate symporter CitS of Klebsiella pneumoniae. Compared to classical hybridoma technology, the in vitro selection systems allow a much better control of the structural integrity of the target protein and allow the use of other protein classes in addition to recombinant antibodies. We also compared the selected DARPins to a Fab fragment previously selected by phage display and demonstrate that different epitopes are recognized, unique to each class of binding molecules. Therefore, the use of several classes of binding molecules will make suitable crystal formation and the determination of their 3D structure more likely.}, address = {Department of Biochemistry, University of Z\"{u}rich, Winterthurerstrasse 190, CH-8057 Z\"{u}rich, Switzerland.}, author = {Huber, Thomas and Steiner, Daniel and R\"{o}thlisberger, Daniela and Pl\"{u}ckthun, Andreas }, citeulike-article-id = {1379682}, doi = {10.1016/j.jsb.2007.01.013}, issn = {1047-8477}, journal = {J Struct Biol}, keywords = {ankryin, crystallization, darpins, membrane, protein, repeat}, month = {February}, posted-at = {2007-06-12 05:17:55}, priority = {2}, title = {In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na(+)-citrate symporter CitS as an example.}, url = {http://dx.doi.org/10.1016/j.jsb.2007.01.013}, year = {2007} }

TY - JOUR ID - citeulike:1379682 L3 - citeulike-article-id:1379682 TI - In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na(+)-citrate symporter CitS as an example. JF - J Struct Biol AD - Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. SN - 1047-8477 N2 - The determination of 3D structures of membrane proteins is still extremely difficult. The co-crystallization with specific binding proteins may be an important aid in this process, as these proteins provide rigid, hydrophilic surfaces for stable protein-protein contacts. Also, the conformational homogeneity of the membrane protein may be increased to obtain crystals suitable for high resolution structures. Here, we describe the efficient generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) as specific binding molecules for membrane proteins. We used both phage display and ribosome display to select DARPins in vitro that are specific for the detergent-solubilized Na(+)-citrate symporter CitS of Klebsiella pneumoniae. Compared to classical hybridoma technology, the in vitro selection systems allow a much better control of the structural integrity of the target protein and allow the use of other protein classes in addition to recombinant antibodies. We also compared the selected DARPins to a Fab fragment previously selected by phage display and demonstrate that different epitopes are recognized, unique to each class of binding molecules. Therefore, the use of several classes of binding molecules will make suitable crystal formation and the determination of their 3D structure more likely. KW - ankryin KW - crystallization KW - darpins KW - membrane KW - protein KW - repeat AU - Huber, Thomas AU - Steiner, Daniel AU - Röthlisberger, Daniela AU - Plückthun, Andreas PY - 2007/02/03/ UR - http://dx.doi.org/10.1016/j.jsb.2007.01.013 ER -