Cloning, expression, and assembly of sericin-like protein.
Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber.
Huang
Jia
J
Department of Biomedical Engineering, Bioengineering Center, Tufts University, Medford, Massachusetts 02155, USA.
Valluzzi
Regina
R
Bini
Elisabetta
E
Vernaglia
Brian
B
Kaplan
David L
DL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D012831:Bombyx; D002942:Circular Dichroism; D004396:Coloring Agents / Q000494:pharmacology; D003224:Congo Red / Q000494:pharmacology; D019476:Insect Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D047030:Sericins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors; D014466:Ultraviolet Rays
D000596:Amino Acids; D004396:Coloring Agents; D019476:Insect Proteins; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D047030:Sericins; D003224:Congo Red