A COOH-terminal peptide confers regiospecific orientation and facilitates atomic force microscopy of an IgG1.
An antibody (IgG1) was designed for oriented adherence to a metal-containing surface. This was achieved by adding a metal-chelating peptide, (CP = His-Trp-His-His-His-Pro), to the COOH-terminus of the heavy chain through genetic engineering. Electroporation of the engineered heavy chain gene into cells expressing the complimentary light chain yielded colonies secreting an intact antibody containing the metal-chelating peptide (IgG1-CP) which had high affinity for a nickel-loaded iminodiacetate column. Purified IgG1-CP was bound to nickel-treated mica and imaged by atomic force microscopy (AFM). Antibody lacking the COOH-terminal metal binding peptide failed to produce discernible AFM images. The AFM images of individual IgG1-CP molecules and their calculated dimensions demonstrated that regiospecific binding and uniform orientation of the antibody was imparted by the peptide. The ability to stably orient macromolecules in their native state to a surface may be used advantageously to visualize them.
Ill
C R
CR
Division of Imaging and Therapeutic Research, Hybritech, Inc., San Diego, California 92121.
Keivens
V M
VM
Hale
J E
JE
Nakamura
K K
KK
Jue
R A
RA
Cheng
S
S
Melcher
E D
ED
Drake
B
B
Smith
M C
MC
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002614:Chelating Agents; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D008670:Metals; D008853:Microscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering
D002614:Chelating Agents; D007074:Immunoglobulin G; D008670:Metals; D009842:Oligopeptides