K J van der Merwe — Page 3

PubMedDate not supplied

Expression of human P450c17 as an export protein in Saccharomyces cerevisiae.

Cytochrome P450c17 (P450c17), together with cytochrome P450c21 (P450c21), plays an important role in progesterone metabolism in the mammalian adrenal cortex. Low levels of expression and the presence of other steroidogenic enzymes in adrenal cortex endoplasmic reticulum (ER) impedes purification and characterisation of wild type as well as mutant forms of the hemoprotein. Heterologous gene expression systems have previously been used successfully to express active P450c17. Heterologous expression can also be used for the preparation of anti-P450c17-IgG. For antibody production larger amounts of pure P450c17 peptide, rather than the active protein, is, however, desirable. If the expressed protein can be affinity tagged and secreted into the medium, isolation and purification will be facilitated. Saccharomyces cerevisiae, YPH259, was transformed with a modified YCplac111 yeast expression-secretion vector (pPRL2). The gene coding for a truncated human P450c17 (signal anchor sequence 1-18 was removed) was inserted, in reading frame, downstream from the leader sequence MF alpha. A histidine tag was incorporated at the C-terminus. The modified yeast expression vector was expressed in yeast, the secreted P450c17-peptide purified by affinity chromatography and identified by immunoblot analysis.

Biological Transport↗

PubMedDate not supplied

Eliciting antibodies in chickens to human dimeric IgA. Removal of factors from human colostrum depressing anti IgA antibody production.

Contrary to expectation chickens did not readily elicit antibodies to IgA dimers when untreated human colostrum was used as antigen. When colostrum was fractionated by means of a column of 8% granulated agarose equilibrated with 10mM phosphate buffer, pH 7.2, a major and a minor fraction were obtained. The major or "1st fraction" consisted of two components with sedimentation coefficients of 10.9 S and 14.1 S, respectively. The minor or "2nd fraction" consisted of components of S values ranging from 2 to 6 and small amounts of 10.9 and 14.1 S material. When chickens were immunized with the "1st fraction" antibodies to dimeric IgA were produced. When the "1st and 2nd fractions" of the column were remixed and used for immunization of chickens, the immune response was as poor as when the chickens were injected with untreated colostrum. An immuno-depressing agent in colostrum was indicated. When rabbits were immunized with clarified human colostrum, antibodies against five antigens were elicited, one of the antigens being dimeric IgA. The immuno-depressing agent is therefore not universal. The purified agent suppressed antibody formation in chickens against the haemocyanin of Jasus lalandii. The "activity" is therefore not specific for IgA and the remaining four antigens in human colostrum. The purified component is a glyco-protein with a hexose content in excess of 10%. The derivatized sugars prepared from it were shown by gas liquid chromatography to be an equimolar mixture of galactose, mannose and fucose. The molecular weight (Mr) of the purified component was found to be 72,000 by sedimentation and diffusion and 80,000 by SDS page using Mr reference standards. The properties of the immuno-suppressor strongly suggest that it is the secretory piece of dimeric IgA.

Animals↗