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Han M.,Huaiyin Normal University | Yu X.,Key Laboratory of Carbohydrate Chemistry and Biotechnology
Bioengineered | Year: 2015

Yeasts are widely used for the production of heterologous proteins. Improving the expression of such proteins is a top priority for pharmaceutical and industrial applications. N-Glycosylation, a common form of protein modification in yeasts, facilitates proper protein folding and secretion. Accordingly, our previous study revealed that the attachment of additional N-glycans to recombinant elastase by introducing an Nglycosylation sequon at suitable locations could stimulate its expression. Interestingly, the sequon Asn-Xaa-Thr is N-glycosylated more efficiently than Asn-Xaa- Ser, so improving the N-glycosylation efficiency via the conversion of Ser to Thr in the sequon would enhance the efficiency of N-glycosylation and increase glycoprotein expression. Recently, the expression level of recombinant elastase was enhanced by this means in our lab. Actually, the modification of N-glycosylation sites can generally be achieved through site-directed mutagenesis; thus, the method described in this report represents a feasible means of improving heterologous protein expression in yeasts. © 2015 Taylor & Francis Group, LLC. Source


Tan Z.,Key Laboratory of Carbohydrate Chemistry and Biotechnology | Lu W.,Key Laboratory of Carbohydrate Chemistry and Biotechnology | Li X.,Jiangnan University | Yang G.,Key Laboratory of Carbohydrate Chemistry and Biotechnology | And 4 more authors.
Journal of Proteome Research | Year: 2014

Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-β1 (TGFβ1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGFβ-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans, and fucosylation were suppressed, and bisecting GlcNAc N-glycans were greatly suppressed. The expression of seven N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology. © 2014 American Chemical Society. Source


Yin X.,Key Laboratory of Carbohydrate Chemistry and Biotechnology | Hu D.,Key Laboratory of Carbohydrate Chemistry and Biotechnology | Li J.-F.,Jiangnan University | He Y.,Key Laboratory of Carbohydrate Chemistry and Biotechnology | And 2 more authors.
PLoS ONE | Year: 2015

The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re- AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152Cwas similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed. © 2015 Yin et al. Source

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