Bacterially produced human B7-1 protein encompassing its complete extracellular domain maintains its costimulatory activity in vitro.
OBJECTIVE
To investigate which of the two immunoglobulin (Ig)-like domains, immunoglobulin variable region homologous domain IgV (hB7-1 IgV), or immunoglobulin constant region homologous domain IgC (hB7-1 IgC) on human B7-1 molecule contain the receptor binding sites, and to evaluate if the B7-1 molecule expressed in bacteria has biological activity.


METHODS
PCR was used to amplify three fragments of hB7-1 IgV, hB7-1 IgC and complete extracellular region of human B7-1 containing both the IgV and IgC domains (hB7-1 IgV + IgC). Three recombinants, pQE9-hB7-1 IgV, pQE9-hB7-1 IgC and pQE9-Hb7-1 (IgV + IgC) were generated by cloning the PCR products into a prokaryote expression plasmid (pQE-9) and were introduced into the host stain M15. The relevant target hexahistidine-tagged proteins were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7-1 proteins and [3H]-TdR incorporation.


RESULTS
Three recombinant proteins of human B7-1, hB7-1 IgV, hB7-1 IgC and hB7-1 (IgV + IgC) were produced and detected in both soluble and inclusive body forms from engineered bacterial cells. With the presence of anti-CD3 antibody, T lymphocytes proliferated when co-stimulated by bacterially produced hB7-1 (IgV + IgC), but not by either hB7-1 IgV or hB7-1 IgC.


CONCLUSIONS
Functional glycoprotein human B7-1 could be produced in bacterial cells. Both extracellular immunoglobulin-like domains are necessary for B7-1 to react with its counter receptors.
Shen
W
W
Institute of Immunopathology, Xi'an Medical University, 205 Scarlet Bird Street, Xi'an 710061, China.
Wang
Y
Y
Geng
Y
Y
Si
L
L
D018122:B7-1 Antigen / Q000096:biosynthesis*; D017252:CD3 Complex / Q000276:immunology; D002455:Cell Division; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D011387:Prokaryotic Cells / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology
D018122:B7-1 Antigen; D017252:CD3 Complex; D007135:Immunoglobulin Variable Region