Targeted protein functionalization using His-tags.
With the impressive growth in gene sequence data that has become available, recombinant proteins represent an increasingly vast source of molecular components, with unique functional and structural properties, for use in biotechnological applications and devices. To facilitate the use, manipulation, and integration of such molecules into devices, a controllable method for their chemical modification was developed. In this approach, a trifunctional labeling reagent first recognizes and binds a His-tag on the target protein's surface. After binding, a photoreactive group on the trifunctional molecule is triggered to create a covalent linkage between the reagent and the target protein. The third moiety on the labeling reagent can be varied to bring unique chemical functionality to the target protein. This approach provides: (1) specificity in that only His-tagged targets are modified, (2) regio-specific control in that the target is modified proximal to the His-tag, the position of which can be varied, and (3) stoichiometric control in that the number modifications is limited by the binding capacity of the His-tag. Two such labeling reagents were designed, synthesized, and used to modify both N- and C-terminally His-tagged versions of the enzyme murine dihydrofolate reductase (mDHFR). The first reagent biotinylated the enzyme,while the second served to attach an oligonucleotide to yield a protein-DNA conjugate. In all cases, modification in this manner brings new functionality to the protein while leaving the enzymatic activity intact. The protein-DNA conjugate was used to specifically immobilize the active enzyme through DNA hybridization onto polystyrene microspheres, a step toward creating a functional protein microarray.
Meredith
Gavin D
GD
Department of Physiology & Biophysics, University of California - Irvine, Irvine, California 92697-4560, USA. Gavin.Meredith@invitrogen.com
Wu
Hayley Y
HY
Allbritton
Nancy L
NL
D004247:DNA / Q000032:analysis / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods*; D006639:Histidine / Q000032:analysis* / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000032:analysis / Q000235:genetics
D016376:Oligonucleotides, Antisense; C033223:polyhistidine; D006639:Histidine; D004247:DNA