Abstract
Herein, we report the extracellular expression of carbohydrate active fusion enzymes in Pichia pastoris. Particularly, CBM29-1-2 from Piromyces equi was separately fused to the N- and C-terminus of galactose 6-oxidase (GaO, D-galactose: oxygen 6-oxidoreductase, EC 1.1.13.9, CAZy family AA5) from Fusarium graminearum, generating CBM29-GaO and GaO-CBM29, respectively. P. pastoris was transformed with expression vectors encoding GaO, CBM29-GaO and GaO-CBM29, and the fusion proteins were expressed in both shake-flask and 2L bioreactor systems. Volumetric production yields and specific GaO activity increased when expression was performed in a bioreactor system compared to shake-flask cultivation. This was observed for both CBM29-GaO and GaO-CBM29, and is consistent with previous reports of GaO expression in P. pastoris (Spadiut et al., 2010; Anasontzis et al., 2014) [1], [2]. Fusion of CBM29 to the C-terminal of GaO (GaO-CBM29) resulted in a stable uniform protein at the expected calculated size (107 kDa) when analyzed with SDS-PAGE. By comparison, the expression of the N-terminal fusion protein (CBM29-GaO) was low, and two truncated versions of CBM29-GaO were coexpressed with the full-sized protein. Despite differences in protein yield, the specific GaO activity on galactose was not affected by CBM29 fusion to either the N- or C-terminus of the enzyme. A detailed description of the catalytic and physiochemical properties of CBM29-GaO and GaO-CBM29 is available in the parent publication (Mollerup et al., 2015) [3].
Highlights
Master / Data in Brief 6 (2016) 176–183 protein yield, the specific GaO activity on galactose was not affected by CBM29 fusion to either the N- or C-terminus of the enzyme
A detailed description of the catalytic and physiochemical properties of CBM29-GaO and GaO-CBM29 is available in the parent publication (Mollerup et al, 2015) [3]
Pichia pastoris transformants encoding CBM29-GaO or GaO-CBM29 for extracellular expression were grown overnight in 300 mL buffered minimal glycerol medium (BMGY (w/v): 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, 1.34% yeast nitrogen base without amino acids (YNB), 4 Â 10À5% biotin, 1% glycerol) at 30 °C with continuous shaking at 250 rpm
Summary
Tables and Figures Through analysis of data from recombinant protein expression Data is analysed and presented in text Recominant expression and purification of fusion proteins constructed by separately appending a family 29 carbohydrate binding module to the N- and Cterminus of galactose oxidase Protein expression in shake-flasks and bioreactor systems and chromatographic methods to purify target proteins from cell culture supernatants Not applicable. These results represent the first production and purification study of galactose oxidase fusions to non-native carbohydrate binding modules, and investigates the impact of CBM positioning on protein recovery. 3. Observation of truncated forms of CBM29-GaO, which co-expressed with the full-sized protein. 4. Isolation of full-sized CBM29-GaO from its truncated versions by ion-exchange chromatography utilizing slight differences in calculated pI values
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