Secretion of a low‐molecular‐weight species of endogenous GRP94 devoid of the KDEL motif during endoplasmic reticulum stress in Chinese hamster ovary cells

Traffic ◽  
2021 ◽  
Author(s):  
Andrew Samy ◽  
Noriko Yamano‐Adachi ◽  
Yuichi Koga ◽  
Takeshi Omasa
Keyword(s):  
Molecular Weight ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Molecular Weight ◽  
Ovary Cells

Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4063-4070
Author(s):  
A J Dorner ◽  
M G Krane ◽  
R J Kaufman
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary ◽  
Heterologous Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Ovary Cells ◽  
Tissue Plasminogen ◽  
Stable Association

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.

scholarly journals Characterisation of Recombinant Human Erythropoietin Obtained from CHOpE—Erythropoietin Producing Strain of Chinese Hamster Ovary Cells

2020 ◽  
Vol 20 (2) ◽  
pp. 116-125
Author(s):  
I. F. Radaeva ◽  
V. A. Ternovoy ◽  
A. O. Sementsova ◽  
N. B. Dumchenko ◽  
E. A. Nechaeva
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
High Molecular Weight ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Producer Strain ◽  
Ovary Cells ◽  
Related Substances

Development and implementation in clinical practice of recombinant human erythropoietins (rhEPOs) remain a priority task today. Additional studies were performed in order to obtain clinical trial authorisation for rhEPO tablets for oral use. The studies were aimed to demonstrate the suitability of the erythropoietin producer strain based on Chinese hamster ovary cells (CHOpE) for the production of rhEPO, and the compliance of the substance characteristics with the requirements for erythropoietin (EPO).The aim of the study was to characterise the rhEPO substance obtained from the CHOpE strain cells in accordance with the requirements for EPO.Materials and methods: the rhEPO substance was obtained by culturing the strain of Chinese hamster ovary cells—CHOpE. The expression construct of the producer strain was evaluated using methods for determination of nucleotide and amino acid sequences. The Sanger method was used to perform sequencing of the nucleotide sequence encoding the human EPO gene. The amino acid sequences of the rhEPO molecule C- and N-termini were determined by the Edman method. The copy number of the EPO gene in CHOpE cells was determined by real-time polymerase chain reaction. The properties of the rhEPO substance were evaluated in accordance with the requirements for EPO. Isoelectric focusing, peptide mapping, and polyacrylamide gel electrophoresis were used for identification of the rhEPO substance. The ratio of isoform composition was determined by capillary electrophoresis. Dimer impurities and high molecular weight related substances were determined by high-pressure liquid chromatography. The content of protein and residual nucleic acids was determined by spectrophotometry. The concentration of the rhEPO substance was assessed by enzyme immunoassay.The results of the study confirmed genetic stability of the CHOpE producer strain and demonstrated identity of N- and C-terminal amino acid sequences of the rhEPO molecule to those of the natural EPO. The CHOpE producer strain was used to obtain a rhEPO substance which is homogenous and does not contain impurities of EPO oligomeric forms. Dimers and high molecular weight related substances account for less than 0.5%. The rhEPO molecular weight ranges from 32 to 38 kDa, and the isoelectric point is within 2.8—4.15. The study identified the peaks of isoforms 1–8, the isoform composition of the rhEPO substance corresponds to that of EPO. It was determined that 1 mol of the substance contains 13.75 mols of sialic acids.Conclusions: the study confirmed the suitability of the CHOpE producer strain for the production of rhEPO. The obtained rhEPO substance meets requirements for EPO. 

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