Direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase.
Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.
Wong
Lu Shin
LS
School of Chemistry and Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Thirlway
Jenny
J
Micklefield
Jason
J
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049409:Luciferases, Firefly / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D040081:Protein Array Analysis / Q000379:methods*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C082662:phosphopantetheinyl transferase; D049409:Luciferases, Firefly; D005982:Glutathione Transferase; D017855:Transferases (Other Substituted Phosphate Groups); D003065:Coenzyme A