Optimization of glycosylation type of recombinant human growth hormone-Fc fusion protein expressed in CHO cells / 中国生物制品学杂志

@#Objective To optimize the expression of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein in CHO cells in order to obtain better glycosylation ratio and lower content of highmannose.Methods CHO cells expressing rhGH-Fc were cultured in a 7 L bioreactor.The glycosylation modifications of rhGH-Fc were adjusted by improving the composition of feeding media(using three commercial media:Gly-1:EX-CELL Glycosylation Adjust,Gly-2:SHEFF-CHO PLUS PG ACF and Gly-3:EfficientFeed C + AGT Supplement & GlycanTune C + Total Feed),and the glycosylation type and proportion of the target proteins were analyzed by mass spectrometry.Results The G0F(main glycosylation types:G0,G1 and G2;F:fucose)of Gly-1,Gly-2 and Gly-3 were 32.89%,58.66% and 33.28%,the G1F were 31.39%,18.03%and 34.90%,and the G2F were 31.39%,18.03% and 34.90%,respectively.Gly-1 and Gly-3 made the target protein contain less G0F while more G2F;Gly-3 feeding scheme-showed less high mannose modification than the other two schemes.Conclusion Gly-1 medium changed the glycosylation modification from G0F to G1F and G2F,while Gly-2 medium changed that from G2F and G1F to G0F.However,Gly-3 medium changed the glycosylation modification from G0F to G1F and G2F,and the contentof high mannose was less than 5%,which may have a better effect on modifying glycosylation type and proportion of the target protein.

The Effect of EOGT-Mediated O-Glcnac Modification on Notch Signaling Pathway and Its Role in Diseases / 中国生物化学与分子生物学报

O-linked-N-acetylglucosamine (O-GlcNAc) modification is a unique post-translational modification that plays a regulatory role in many cellular processes, such as transcription, intracellular signaling, endocytosis, and protein stability. Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER) resident protein which can glycosylate the residues of Ser or Thr of secreted or membrane (transmembrane) glycoproteins containing EGF domain. Notch signaling pathway is involved in cell-to-cell communication which regulates cell biological processes through interactions between adjacent cells. To date, EOGT-mediated O-GlcNAc modification has been found to be involved in many human diseases, and shown significant relation with Notch signaling pathway. However, the specific molecular mechanisms have not been fully elucidated. In this review, we briefly introduce recent studies regarding to the roles of EOGT-mediated O-GlcNAc modification and its correlation with Notch signaling pathway in human diseases.

Identification of the glycosylation sites of Opsin3 and its glycosylation modification function / 生物工程学报

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

N-glycosylation modification of heat shock protein gp96 affects its immunological function / 生物工程学报

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

Applications of glycosyltransferases in the improvement of druggability of natural products / 中国药科大学学报

Natural products are one of the most important sources for druggable lead compounds given their diverse structures and activities.Nevertheless,very few of them can be directly developed to drugs.The undesirable druggability of natural products result from their weaker activity,lower aqueous solubility and poorer stability.Since improving druggability by chemical modifications is restricted by the limited access to the complex structures of natural products,enzymatic modification has gradually become one of important tools for the structural modification of natural products.Meanwhile,since enzymatic glycosylation by glycosyltransferases has shown certain advantages such as excellent regio-and stereo-selectivity,high catalytic efficiency and mild conditions.It has been a promising route for the improvement of druggability for natural products.In the present review,we summarize different glycosyltransferases and their application for structural modification of natural products as well as the challenges issues involved in the glycosylation,which provides a general perspective on the enzymatic modification of natural products for the improvement of their draggability.

Expression and purification of Ebola virus trimeric glycoprotein based on novel glycoengineered Pichia pastoris / 军事医学

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.