Expression, cross-linking, and characterization of recombinant chitin binding resilin.
Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein--carbohydrate composite material. We describe the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (K(d), B(max)) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier transform infrared spectroscopy, circular dichroism, and atomic force microscopy with peroxidase cross-linked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine cross-links. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.
Qin
Guokui
G
Department of Biomedical Engineering, 4 Colby Street, Tufts University, Medford, Massachusetts 02155, USA.
Lapidot
Shaul
S
Numata
Keiji
K
Hu
Xiao
X
Meirovitch
Sigal
S
Dekel
Mara
M
Podoler
Itai
I
Shoseyov
Oded
O
Kaplan
David L
DL
D000595:Amino Acid Sequence; D000818:Animals; D002686:Chitin / Q000737:chemistry* / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics; D004331:Drosophila melanogaster; D004548:Elasticity; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics
D029721:Drosophila Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins; D002686:Chitin; C030951:resilin