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Characterization of Human Bone Alkaline Phosphatase in Pichia PastorisA soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.
Document ID
19990079357
Acquisition Source
Marshall Space Flight Center
Document Type
Reprint (Version printed in journal)
Authors
Malone, Christine C.
(NASA Marshall Space Flight Center Huntsville, AL United States)
Ciszak, Eva
(NASA Marshall Space Flight Center Huntsville, AL United States)
Karr, Laurel J.
(NASA Marshall Space Flight Center Huntsville, AL United States)
Date Acquired
August 19, 2013
Publication Date
January 1, 1999
Subject Category
Aerospace Medicine
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.

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