Microbial expression of proteins containing long repetitive Arg-Gly-Asp cell adhesive motifs created by overlap elongation PCR.
We developed a novel method for creating repetitive DNA libraries using overlap elongation PCR, and prepared a DNA library encoding repetitive Arg-Gly-Asp (RGD) cell adhesive motifs. We obtained various length DNAs encoding repetitive RGD from a short monomer DNA (18 bp) after a thermal cyclic reaction without a DNA template for amplification, and isolated DNAs encoding 2, 21, and 43 repeats of the RGD motif. We cloned these DNAs into a protein expression vector and overexpressed them as thioredoxin fusion proteins: RGD2, RGD21, and RGD43, respectively. The solubility of RGD43 in water was low and it formed a fibrous precipitate in water. Scanning electron microscopy revealed that RGD43 formed a branched 3D-network structure in the solid state. To evaluate the function of the cell adhesive motifs in RGD43, mouse fibroblast cells were cultivated on the RGD43 scaffold. The fibroblast cells adhered to the RGD43 scaffold and extended long filopodia.
Kurihara
Hiroyuki
H
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Shinkai
Masashige
M
Nagamune
Teruyuki
T
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D051379:Mice; D008855:Microscopy, Electron, Scanning; D041681:NIH 3T3 Cells; D009842:Oligopeptides / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D020449:Repetitive Sequences, Amino Acid
D017931:DNA Primers; D004274:DNA, Recombinant; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C047981:arginyl-glycyl-aspartic acid