Main purpose of this study was to increase protein expression in Chinese hamster ovary (CHO) cells by improving protein secretion of translated proteins. The goal was to find signal peptides from the screening of signal peptide libraries for improvement of protein secretion using a CHO-cell express selection system. Biopharmaceutical products, proteins such as monoclonal antibodies (mAbs), are most commonly produced using mammalian expression systems such as the expression in CHO cells. The posttranslational modifications of the proteins being expressed in CHO cells are similar to the expressional modifications in human cells, why the CHO cells are suitable for production of proteins used for human therapy. The expression of proteins in the cell is a complex mechanism, fundamentally depending on the DNA sequences in the cell nucleus. Secretion of translated proteins has been showed to be a bottleneck when improving expression. Secretion is initiated by the signal peptide, a n-terminal prolongation of the protein that is recognized by a signal recognition particle (SRP) when being translated by the ribosome. The sequence and structure of the signal peptide has been proved to affect secretion and altering the signal peptide could improve secretion even when changing signal peptide between different species. Designing variants of the signal peptides and analyzing protein expression might lead to improvements of the construct design and more protein produced from the cells, which would save time, money and material for the producer. To construct plasmids containing the gene of interest (GOI) and different signal peptides, several gene cloning methods were used. The plasmids were amplified using Escherichia coli (E. coli) transformation. The constructs were expressed by transfection into the CHO cell genome, and expression were analyzed using flow cytometry. When analyzing expression of a Fc-fusion protein with 5 different signal peptides, the signal peptide Azurocidin is the one showing highest expression levels in this study. In addition, IgG kapa and Albumin signal peptides did not show as high protein expression levels, even if they were better than the L1d and H5b signal peptides. Since signal peptides are exchangeable between proteins and species, it might be that Azurocidin is improving secretion and protein expression with other proteins than Fc-fusion proteins which would be an interesting aspect for further studies. When altering signal peptides with library sequences, the experimental challenges were crucial for the protein expression results and due to these issues, no library sequence could be seen to conquer others when it comes to protein expression levels. Transfection and cultivation procedures needs to be studied and improved before being able to draw conclusions about which signal peptide library sequences that might improve secretion and increase the protein expression.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:liu-150364 |
| Date | January 2018 |
| Creators | Strannermyr, Malin |
| Publisher | Linköpings universitet, Teknisk biologi |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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