Tissue kallikrein is a serine protease involved in the generation of kinins in kidneys, colon, salivary glands, pancreas and blood vessels which have vasodilator roles and influence ion transport. Abnormal renal synthesis and urinary excretion of tissue kallikrein have been linked to diabetes and hypertension. This studys objective was to produce mAbs against native forms of prokallikrein and active kallikrein in order to study the expression of tissue kallikrein in different cell culture conditions and patient samples.
A full-length cDNA encoding human tissue kallikrein (hK1) from a human colon carcinoma cell line (T84) was cloned into mammalian expression vector pcDNA5/FRT/V5-His and stably transfected into HEK-293. The transfected HEK-293 cells were adapted to grow in serum-free suspension culture. These cells synthesised and released inactive prokallikrein into the medium. Prokallikrein was activated by treatment with trypsin or thermolysin. Recombinant hK1 was identified by Western blot analysis with a band of approximately 50 kDa using a rabbit anti-hK1 antibody. Transfected prokallikrein in HEK-293 cells were localised to the cytoplasm with a granular distribution by immunostaining with anti-his tag mAb and rabbit anti-hK1 antibody. Prokallikrein and active kallikrein were purified and used to immunise mice. Following lymphocyte and myeloma fusion, resultant hybridomas were screened and a panel of mAbs selected. In sandwich ELISA, all mAbs recognised transfected prokallikrein and active kallikrein in HEK-293 cells. In immunofluorescence microscopy, these mAbs recognised transfected and endogenous hK1 in the cytoplasm of HEK-293 cells, showing the same localisation as the rabbit anti-hK1 antibody.
Following further characterisation, these new mAbs against native hK1 will be useful tools to study hK1 expression and function.
01 - 05 Apr 2006
European Society of Endocrinology