Purification and characterization of human cell--cell adhesion molecule 1 (C-CAM1) expressed in insect cells.
The cell--cell adhesion molecule 1 (C-CAM1) plays an important role as a tumor suppressor for prostate cancer. Decreased expression of C-CAM1 was detected in prostate, breast, and colon carcinoma. Reexpression of C-CAM1 in prostate and breast cancer cell lines was able to suppress tumorigenicity in vivo. These observations suggest that C-CAM1 may be used as a marker for cancer detection or diagnosis. To generate monoclonal antibodies specific to C-CAM1, we have overexpressed full-length human C-CAM1 in Sf9 cells using a baculovirus expression system. The protein was purified 104-fold using nickel affinity chromatography. About 0.4 mg purified C-CAM1 was obtained from 200 mg of infected cells. When the purified protein was digested with peptidyl-N-glycosidase, the apparent mobility of the protein on SDS--PAGE changed from 90 to 58 kDa, which is close to the molecular weight predicted from the cloned cDNA sequence. This observation suggests that C-CAM1 was glycosylated on asparagine residues when expressed in Sf9 cells. Western blotting and internal protein sequencing analysis confirmed that the purified protein is human C-CAM1. Biochemical and functional assays indicate that this protein expressed in Sf9 cells displays characteristics similar to those of native protein, including adhesion function and glycosylation modification. Using this protocol, sufficient quantity of this protein can be produced with purity suitable for monoclonal antibody generation and biochemical study.
Phan
D
D
Cold Spring Harbor Laboratory, USA.
Han
E
E
Birrell
G
G
Bonnal
S
S
Duggan
L
L
Esumi
N
N
Gutstein
H
H
Li
R
R
Lopato
S
S
Manogaran
A
A
Pollak
E S
ES
Ray
A
A
Reddi
P P
PP
Reichert
A S
AS
Struffi
P
P
Tiscornia
G
G
Ximenez-Fyvie
L A
LA
Zhang
H
H
Lin
S H
SH
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D048429:Cell Size; D002846:Chromatography, Affinity; D006031:Glycosylation; D006801:Humans; D009532:Nickel / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism / Q000821:virology
D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; D009532:Nickel; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000251:Adenosine Triphosphatases