scholarly journals Recombinant human interferon-γ. Differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture

1990 ◽  
Vol 272 (2) ◽  
pp. 333-337 ◽  
Author(s):  
E M Curling ◽  
P M Hayter ◽  
A J Baines ◽  
A T Bull ◽  
K Gull ◽  
...  
Keyword(s):  
Batch Culture ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Interferon Γ ◽  
Proteolytic Processing ◽  
Human Interferon ◽  
Oxygen Control ◽  
Ifn Gamma ◽  
Sds Page ◽  
Dissolved Oxygen Control

Recombinant human interferon-gamma (Hu-IFN-gamma) produced by Chinese-hamster ovary (CHO) cells was analysed by immunoprecipitation and SDS/PAGE. Up to twelve molecular-mass variants were secreted by this cell line. Three variants were recovered after enzymic removal of all N-linked oligosaccharides or when glycosylation was inhibited by tunicamycin. The presence of three polypeptide forms rather than a single form suggested that proteolytic cleavage had occurred at two sites in both the glycosylated and non-glycosylated forms. Proteolytically cleaved IFN-gamma was more prevalent in cell lysates than in the secreted glycoprotein. In common with naturally produced IFN-gamma, both fully glycosylated IFN-gamma (asparagine residues 28 and 100 occupied) and partially glycosylated product (thought to be substituted at position Asn28) were secreted. This was deduced from the Mr of the glycosylated products and the relative amounts of sialic acid expressed by each variant. In contrast with naturally produced IFN-gamma, non-glycosylated IFN-gamma was also secreted by the transfected CHO cells. When the cells were grown in batch culture in serum-free medium under pH and dissolved-oxygen control, the proportion of non-glycosylated IFN-gamma increased from 3 to 5% after 3 h, to 30% of the total IFN-gamma present after 195 h. This change in the proportion of glycosylated protein produced was not seen when metabolically labelled IFN-gamma was incubated for 96 h with cell-free supernatant from actively growing CHO cells. This implied that an alteration in intracellular glycosylation was occurring rather than a degradation of oligosaccharide side chains after secretion. The decrease in IFN-gamma glycosylation was independent of the glucose concentration in the culture medium, but could be related to specific growth and IFN-gamma production rates, as these declined steadily after 50 h of culture, in line with the increased production of non-glycosylated IFN-gamma.

N-glycans of recombinant human interferon-? change during batch culture of chinese hamster ovary cells

1995 ◽  
Vol 48 (6) ◽  
pp. 639-648 ◽  
Author(s):  
Andrew D. Hooker ◽  
Merlin H. Goldman ◽  
Nicola H. Markham ◽  
David C. James ◽  
Andrew P. Ison ◽  
...  
Keyword(s):  
Batch Culture ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Interferon ◽  
Ovary Cells

scholarly journals Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-γ

1994 ◽  
Vol 303 (3) ◽  
pp. 831-840 ◽  
Author(s):  
T Sareneva ◽  
J Pirhonen ◽  
K Cantell ◽  
N Kalkkinen ◽  
I Julkunen
Keyword(s):  
Interferon Γ ◽  
Glycosylation Site ◽  
Biologically Active ◽  
Human Interferon ◽  
Mutant Form ◽  
Ifn Gamma ◽  
Glycosylation Sites ◽  
Mutant Forms ◽  
Kinetics Of

Human interferon-gamma (IFN-gamma) is a secretory glycoprotein, which has two potential N-linked glycosylation sites at positions Asn-25 and Asn-97 of its 143 amino acid long mature polypeptide chain. In order to understand the role of glycan residues in the synthesis and secretion of human IFN-gamma, both or either one of the potential N-linked glycosylation sites were mutated to Gln. The mutant and the wild-type (Wt) polypeptides were expressed in insect cells using a baculovirus vector. Elimination of the N-glycosylation site at position Asn-97 (N97Q) resulted in secreted protein yields of 70-90% as compared with the Wt production, whereas only 10-25% (N25Q) and 1-10% (N25Q,N97Q) levels of protein production was observed when the first or both sites were mutated, respectively. Although there was a difference between extracellular levels of produced protein, the kinetics of secretion was similar for all different IFN-gamma molecules. The Wt and the N-glycosylation site mutants were all secreted as dimers. The formation of biologically active dimers was more efficient for IFN-gamma polypeptides that had the intact glycosylation site at Asn-25 as compared with the other two mutant forms of IFN-gamma. The extent of dimerization correlated well with the observed secretion. The specific antiviral activity was of the same order (1 x 10(7) i.u./mg of protein) for the glycosylated IFN-gamma molecules, whereas it was slightly lower (0.5 x 10(7) i.u./mg of protein) for the unglycosylated mutant form.

scholarly journals Optimization of human interferon beta protein expression in Chinese hamster ovary cells

2021 ◽  
Vol 23 (2) ◽  
pp. 68-75
Author(s):  
Nasrin Xodadadi ◽  
Alireza Saeidinia ◽  
Mehdi Zeinoddini ◽  
Rasoul Khalilzadeh
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Interferon Beta ◽  
Recombinant Dna ◽  
Chinese Hamster Ovary Cells ◽  
Active Form ◽  
Chinese Hamster ◽  
Human Interferon

Background and aims: Human interferon beta-1a (hIFNβ-1a) is a 22.5-kDa glycoprotein used to treat diseases such as multiple sclerosis (MS). Because of appropriate post-translation modifications, protein isolation, and lack of toxicity in Chinese hamster ovary (CHO) cells, we cloned hIFNβ-1a encoding sequence into these cells by recombinant DNA technology to achieve stable expression of this recombinant protein. Methods: The hIFNβ-1a encoding sequence was designed based on the CHO cells’ codon usage and the Gene Bank data, and then syntactically constructed in the pUC57 vector. After confirmation, the synthesized sequence was cloned into the pcDNA3.1 expression vector by using EcoRI and XhoI sites via Escherichia coli DH5α competent cells. Then, the recombinant vector pcDNA-hHIFNβ1a was linearized by BglII and transfected into the CHO cells using lipofectamine. The transfected cells were proliferated and screened by gentamicin. Certain concentrations of zinc sulfate, DMSO, and glycerol were used to enhance protein expression. Finally, the recombinant protein expression was qualitatively evaluated using different techniques. Results: The hIFNβ1a integrity was confirmed by DNA sequencing and specific software. The construction and sub-cloning of hIFNβ1a-pcDNA3.1 in E. coli were confirmed by colony-PCR with specific primers and restriction enzyme mapping. The screening of transfected CHO cells was performed using gentamicin. The protein expression was confirmed by RT-PCR, MTT assay, SDS-PAGE, and Western blot. Comparison of the optimized and control samples demonstrated that chemical treatment enhanced the protein expression. Conclusion: We achieved the stable clones of CHO cells expressing the active form of human interferon beta.

scholarly journals N-glycosylation of human interferon-γ: glycans at Asn-25 are critical for protease resistance

1995 ◽  
Vol 308 (1) ◽  
pp. 9-14 ◽  
Author(s):  
T Sareneva ◽  
J Pirhonen ◽  
K Cantell ◽  
I Julkunen
Keyword(s):  
Biological Activities ◽  
Interferon Γ ◽  
Glycosylation Site ◽  
Cathepsin G ◽  
Human Interferon ◽  
Globular Structure ◽  
Ifn Gamma ◽  
Protease Resistance ◽  
Glycosylation Sites ◽  
Defective Mutant

Human interferon-gamma (IFN-gamma) is a secretory, dimeric glycoprotein that forms a compact globular structure with potential N-linked glycosylation sites at Asn-25 and Asn-97 on the surface of the dimer. In natural leucocyte IFN-gamma (nIFN-gamma), 52%, 39% and 9% of the monomers are core-glycosylated in two, one or none of the potential N-glycosylation sites respectively. Chemical cross-linking of nIFN-gamma with glutaraldehyde revealed that 4, 3, 2 or 1 glycosylation sites occupied 28%, 40%, 26% and 6% of the dimers respectively. In baculovirus-produced wild-type (Wt) and N-linked glycosylation site-defective mutant (N25Q or N97Q, Asn-25 or Asn-97 substituted by Gln) IFN-gamma proteins, the extent of core glycosylation of monomers reflected the glycan composition of dimers. This suggests that dimers are formed randomly and independently of glycosylation. The glycan residues of IFN-gamma, especially at Asn-25, play an important role in protease resistance. Unglycosylated recombinant IFN-gamma proteins (from Escherichia coli and baculovirus) and N25Q IFN-gamma were sensitive to crude granulocyte protease, purified elastase, cathepsin G and plasmin degradation. Fully glycosylated nIFN-gamma and baculovirus Wt and N97Q IFN-gamma showed full or partial resistance to these proteases. These results emphasize the importance of glycan residues, especially at Asn-25, in the proteolytic stability of human IFN-gamma. Whether the differential glycosylation of n- and recombinant IFN-gamma (rIFN-gamma) is reflected in their biological activities in tissues or their clinical applicability is not known.

scholarly journals Isolation and initial characterization of the mammalian midbody.

1982 ◽  
Vol 94 (3) ◽  
pp. 654-661 ◽  
Author(s):  
J M Mullins ◽  
J R McIntosh
Keyword(s):  
Cho Cells ◽  
Total Protein ◽  
Protein Extraction ◽  
Chinese Hamster ◽  
Protein Composition ◽  
Dense Matrix ◽  
Mitotic Spindles ◽  
Sds Page ◽  
The Matrix

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.

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