An Approach to the Removal of Yeast Specific O-Linked Oligo-Mannoses from Human Midkine Expressed in Pichia pastoris Using Site-Specific Mutagenesis

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-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.

Heterologous protein production using the Pichia pastoris expression system

“…In a P. pastoris strain producing human midkine, a heparin-binding protein, three of the threonine residues involved in O-glycosylation were replaced with three alanine residues by site-specific mutagenesis. The resultant human midkine recombinant protein contained no mannose residues and promoted proliferation of CHO cells as well as the native protein, despite the alteration to the amino acid sequence [2].…”

“…The immunogenicity of Pichia-derived proteins is an issue that has attracted interest in the literature with regard to humanizing the glycosylation patterns [2,24]. One group of researchers disrupted the PNO1 gene of P. pastoris, resulting in a reduction of N -liked glycosylation of a recombinant human antithrombin.…”

Characterization of Partially Truncated Human Midkine Expressed inPichia pastoris

“…The expression level was not very high, at most 0.1 g W l in high cell density fermentation, and more than 50z of the rh-midkine secreted in the medium suŠered from yeast-speciˆc mannosylation. 6) It is not easy to isolate the non-mannosylated authentic midkine e‹ciently, especially from the rh-midkine that contains only one molecule of mannose. When the a factor prepro sequence was used for the secretory expression of rh-midkine, a large amount of expression was observed in shake-‰ask cultures (about 30 mg W l medium).…”

Crystallization of Antiangiogenic Kringle V Derived from Human Apolipoprotein A: Crystallization Applied to Purification and Formulation

“…These mannosylations normally occur on threonine or serine residues, resulting in the generation of highmolecular-weight forms on mass spectra, as was observed in this study, in conjunction with the mass spectrum of kringle V. [25][26][27][28][29] These glycosylated derivatives might cause several problems in the body, including immunogenicity. Therefore, these derivatives must be completely removed in clinical situations.…”