In the catalytic cycle of the glycine cleavage system (GCS), the major physiological pathway for glycine degradation, the lipoic acid-containing hydrogen carrier protein (H-protein) plays a pivotal role as a mobile carrier of reaction intermediates. and as a regulator for glycine decarboxylase (P-protein). Defects in the GCS lead to the accumulation of glycine in all body tissues, resulting in the genetic disease glycine encephalopathy (GE). Defects in P-protein lead to more than 80% of reported cases of GE, therefore. routine biochemical analysis only tests P-protein for activity. Unlike other amino acid decarboxylases, P-protein is itself inactive, and requires H-protein for biochemical activity. Currently. researchers use H-protein purified from chicken liver extracts for the P-protein activity assay. However. extraction and purification of chicken H-protein is laborious. costly. and has poor yield, making the expression and purification of H-protein from an alternate source desirable. We have overexpressed recombinant human H-protein in the methylotropic yeast Pichia pastoris by utilizing the inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae a-mating factor secretion signal. Enhanced green fluorescent protein (EGFP) was used as a fusion partner to aid detection of H-protein during expression and purification. Secreted H-protein was detected from 24-96 hours post induction, and constituted the major protein species in the culture medium. H-protein was purified to apparent homogeneity in a single step using nickel-chelating affinity chromatography, and lipoylated using lipoate protein ligase from E. coli. Functional analysis of holo-H-protein using the NAD+ lipoamide dehydrogenase assay demonstrated biochemical activity with the artificial substrate, suggesting that human H-protein produced in P. pastoris may be an appropriate replacement for the chicken H-protein currently used in the biochemical diagnosis of GE.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/1893 |
| Date | 24 November 2009 |
| Creators | Zay, Agnes |
| Contributors | Choy, Francis Y. M. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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