An approach to the removal of yeast specific O-linked oligo-mannoses from human midkine expressed in Pichia pastoris using site-specific mutagenesis.
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-terminal 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.
Asami
Y
Y
Meiji Cell Technology Center, Meiji Milk Products Co., Ltd., Naruda, Odawara, Kanagawa 250-0862, Japan.
Nagano
H
H
Ikematsu
S
S
Murasugi
A
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D016207:Cytokines / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008358:Mannose / Q000737:chemistry*; D000078792:Midkine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics*; D010843:Pichia / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry; D015183:Restriction Mapping
D002352:Carrier Proteins; D016207:Cytokines; D009414:Nerve Growth Factors; D011108:Polymers; D000078792:Midkine; D008358:Mannose