Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell-free system.
Biotechnol Bioeng
; 115(5): 1253-1264, 2018 05.
Article
in En
| MEDLINE
| ID: mdl-29384203
ABSTRACT
Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have â¼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biotechnology
/
Recombinant Proteins
/
Cell-Free System
/
Erythropoietin
/
Microsomes
Limits:
Animals
/
Humans
Language:
En
Journal:
Biotechnol Bioeng
Year:
2018
Document type:
Article