In vitro cell-free synthesis of bullous pemphigoid polypeptide

We investigated the genetic expression of bullous pemphigoid (BP) antigen (230 kD) synthesized by Pam cells using an in vitro cell-free translation assay followed by immunoprecipitation. Prior to the study, we showed that (1) the 230 kD polypeptide does not undergo further processing after it is produced by cells in the short pulse experiment; (2) the 230 kD polypeptide is not degraded at least within the 18 h of the chase experiment; and (3) with tunicamycin treatment the underglycosylated BP antigen still interacts specifically with BP serum. The polypeptide of molecular size slightly greater than 230 kD was identified in the translated proteins directed by ribonucleic acid (RNA) isolated from Pam cells. The size of the mRNA was estimated to be approximately 34S-40S (7.7-10.9 kilobase) using the fractionation method with sucrose density gradient ultracentrifugation of RNA. These results indicate that bullous pemphigoid antigen (230 kD) is a primarily translated product, genetically expressed by Pam cells.