Atypical Processing of Amyloid Precursor Fusion Protein by Proteolytic Activity in Pichia pastoris,

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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Enhancing the Production of Fc Fusion Protein in Fed-Batch Fermentation of Pichia pastoris by Design of Experiments

Biotechnology Progress ◽

10.1021/bp0603199 ◽

2008 ◽

Vol 23 (3) ◽

pp. 621-625 ◽

Cited By ~ 20

Author(s):

Henry Lin ◽

Tina Kim ◽

Fei Xiong ◽

Xiaoming Yang

Keyword(s):

Pichia Pastoris ◽

Fusion Protein ◽

Design Of Experiments ◽

Batch Fermentation ◽

Fed Batch ◽

Fc Fusion Protein ◽

Fed Batch Fermentation

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In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris

Biologia ◽

10.2478/s11756-012-0056-3 ◽

2012 ◽

Vol 67 (4) ◽

Cited By ~ 1

Author(s):

Jiayun Qiao ◽

Yunhe Cao

Keyword(s):

Bacillus Subtilis ◽

Pichia Pastoris ◽

Fusion Protein ◽

Fusion Expression ◽

Chimeric Genes ◽

Fusion Enzyme ◽

Fusion Enzymes ◽

Aspergillus Sulphureus

AbstractTwo chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Kn higher kcat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.

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