Chemoselective attachment of biologically active proteins to surfaces by expressed protein ligation and its application for "protein chip" fabrication.
The present work describes a general method for the selective attachment of proteins to solid surfaces through their C-termini that can be used for the efficient creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal alpha-thioester and a modified surface containing N-terminal Cys residues. alpha-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing engineered inteins. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain using a protein microarrayer.
Camarero
Julio A
JA
Chemical Biology and Nuclear Sciences Division and Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA. camarero1@llnl.gov
Kwon
Youngeun
Y
Coleman
Matthew A
MA
D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties; D018909:src Homology Domains
C417561:Alexa 488 hydrazide; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D006834:Hydrazines; D008164:Luminescent Proteins; D010455:Peptides; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; C011083:polyproline; D011092:Polyethylene Glycols; D003545:Cysteine