The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
An Approach to the Removal of Yeast Specific O-Linked Oligo-Mannoses from Human Midkine Expressed in Pichia pastoris Using Site-Specific Mutagenesis
Yukio AsamiHitomi NaganoShinya IkematsuAkira Murasugi
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2000 Volume 128 Issue 5 Pages 823-826

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Abstract

Human midkine is expressed and secreted in the medium under the control of an AOX1 gene promoter in Pichia pastoris using its own secretion signal. The midkine precursor is properly processed to yield the correct amino-terminus of mature midkine. However, more than half of the product receives yeast specific mannosylations. The sites for the mannosylations were determined to be the three threonine residues in the carboxy-ter-minal region of human midkine. In order to obtain non-mannosylated midkine, alanine residues were substituted for the three threonine residues by site specific mutagenesis. HPLC and mass spectrometry confirmed that the mutant midkine contained almost no mannose residues. Despite the amino acid substitutions in the carboxy-terminal region, mutant human midkine, promoted CHO cell proliferation as well as normal midkine.

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© The Japanese Biochemical Society
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