Production of glycosylated heparin-binding EGF-like growth factor in HeLa cells using vaccinia virus.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a 22-kDa, O-glycosylated protein. Because recombinant expression systems permitting a detailed analysis of the functional significance of HB-EGF glycosylation have not been described, a recombinant vaccinia virus designed to express HB-EGF was generated by homologous recombination of an intermediate plasmid vector carrying the HB-EGF cDNA and the genome of vaccinia virus and was used to infect HeLa cells. Production of immunoreactive HB-EGF was confirmed by immunofluorescence and radioimmunoprecipitation analysis. Furthermore, the expressed protein was shown to be a secreted, biologically active protein by radioreceptor and DNA synthesis assays of HeLa cell conditioned medium. The recombinant protein was purified from the conditioned medium using heparin-affinity fast protein liquid chromatography followed by C4 reverse-phase high-performance liquid chromatography (RP-HPLC). SDS-PAGE and Western blotting of the RP-HPLC-purified product showed an immunoreactive HB-EGF protein of approximately 22 kDa that was decreased to a 14-kDa protein by treatment with O-glycanase. Amino acid sequencing revealed an N-terminus that was characteristic of native, glycosylated HB-EGF. Interestingly, a Thr residue that is a putative site of O-linked glycosylation failed to be resolved. This system provides a valuable method for evaluating the role of glycosylation in HB-EGF function(s) as well as addressing other questions concerning HB-EGF structure-function relationships.
Davis
K M
KM
Molecular, Cellular, and Developmental Biology Program, The Ohio State University and Children's Hospital, Columbus, Ohio, 43205, USA.
Brigstock
D R
DR
Johnson
P R
PR
Crissman-Combs
M A
MA
McCarthy
D W
DW
Downing
M T
MT
Besner
G E
GE
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006367:HeLa Cells; D006493:Heparin / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D014162:Transfection / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics*
D017931:DNA Primers; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D006493:Heparin; D006596:Hexosaminidases; C060638:glycopeptide alpha-N-acetylgalactosaminidase