Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.
We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.
Bäckström
Malin
M
Department of Medical Biochemistry, Göteborg University, Gothenburg, Sweden.
Link
Thomas
T
Olson
Fredrik J
FJ
Karlsson
Hasse
H
Graham
Rosalind
R
Picco
Gianfranco
G
Burchell
Joy
J
Taylor-Papadimitriou
Joyce
J
Noll
Thomas
T
Hansson
Gunnar C
GC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001943:Breast Neoplasms / Q000378:metabolism*; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002277:Carcinoma / Q000378:metabolism; D045744:Cell Line, Tumor; D006224:Cricetinae; D005260:Female; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D018396:Mucin-1; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins