Construction and characterization of phage-displayed leukocyte surface molecule CD99.
The phage display technique has been described for the production of various recombinant molecules. In the present report, we used this technique to display a leukocyte surface molecule, CD99. PCR subcloning of CD99 cDNA from the mammalian expression vector pCDM8 to the phagemid expression vector pComb3HSS was performed. The resulting phagemid, pComb3H-CD99, was transformed into Escherichia coli XL-1 Blue. CD99 was displayed on the phage particles following infection of the transformed E. coli with the filamentous phage VCSM13. Using sandwich ELISA, the filamentous phage-displayed CD99 was captured by a CD99 monoclonal antibody (mAb) then detected with anti-M13 conjugated to horseradish peroxidase, confirming that the CD99 molecule was displayed on the phage particles. The CD99-phages inhibited induction of Jurkat cell aggregation by CD99 mAb MT99/1. Proper folding of the displayed CD99 bioactive domain was inferred from this finding. Our results demonstrate that the phage display technique can be applied to the generation of full-length CD99 molecules. The phage carrying this cell surface protein will be useful for identification of its counter receptor or ligand.
Tayapiwatana
C
C
Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. asimi002@chiangmai.ac.th
Kasinrerk
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W
D000071443:12E7 Antigen; D015703:Antigens, CD / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D019151:Peptide Library
D000071443:12E7 Antigen; D015703:Antigens, CD; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; D019151:Peptide Library