Recombinant Proteins

Recombinant Proteins
 

Recombinant proteins eukaryotic expression system
Bacteria and eukaryotic expression system is the most common recombinant proteins express host. In eukaryotic protein function research, as well as in drug development, eukaryotic expression system has its obvious advantages, such as protein folding, assembly, and post-translational modifications [1, 2].Chinese Hamster ovary (CHO) cells and Human embryonic kidney (HEK) cells are widely used in the development and production of common protein and therapeutic recombinant protein drugs.
 
CHO cell
Chinese Hamster ovary (CHO) cells are an epithelial cells line derived from the ovary of Chinese hamster. Since the original CHO cell line was described in 1956, many variants of the cell line have been developed for various purposes. CHO-K1 was generated from a single clone of CHO cells,which was mutagenized with ethyl methanesulfonate to generate a cell line lacking DHFR activity, referred to as CHO-DXB11 (also referred to as CHO-DUKX)[3].
 
At present, the research on CHO cells has been in-depth, and the genome sequencing of female Chinese hamster (Cricetulus griseus), cho-k1, DG44 and cho-s has been completed [4-6]. These basic researches will greatly promote its application in scientific research and bio-pharmaceutical industry [7].
 
HEK cells
Human embryonic kidney cells 293, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. HEK 293 cells have been widely used in cell biology research for many years, because of their reliable growth and propensity for transfection.

HEK 293 cells were generated by transfection of Adenovirus 5 DNA to normal human embryonic kidney cells [8], which contains the 5’ -end sequence of Adenovirus 5 DNA [9].

An important variant of the cell line is 293 T cell line, which contains of SV40 Large T antigen, allows the replication of plasmid containing SV40 duplicate starting point, which enable the amplification of transfection of plasmid and prolong the expression time of the desired gene product [8, 10]. 293FT is a cell clone isolated from 293T cell lines, with higher growth rate and transfection efficiency.

Recombinant protein expression system of Antagen (Beijing) is efficient, fast and stable, which is based on unique technology research and development team in the United States, can significantly increase the cell amount of protein expression and production rate, and significantly shorten the development of stable cell strain (below) about half of the time, at the same time can provide labels removed, virus inactivation, serum-free suspension culture and so on the many kinds of special requirements.

Antagen (Beijing) will provide flexible and quality services for customers, and meet the requirements of pharmaceutical supervision of global biopharmaceutical enterprises.
 
Advantages of Antagen (Beijing)’s recombinant protein expression system

  • Rapid, highly-stringent screening: screening the high expression of stable strains; obtain stable and high expression cell lines after 4~5 months, while conventional methods need 9~12 months.

  • Efficiently: Based on super promoter and super secretion signal peptide, it is easy to reach more than 30 p/c/d/, while traditional methods need to use drug screening more than 9 ~ 11 rounds to achieve the same level.

  • Stable expression: the AGE on the expression plasmid can inhibit the gene silencing of chromosome, and make the cell lines could expressed stably for a long time.

  • GS screening: there is glutamine synthetase (GS) gene on the expression plasmid, as an alternative selection marker. So MSX screening can be used to further enhance the expression of protein yield;

  • High yield rate: the yield of monoclonal cell lines flask can reach 1.5 ~ 2 g/l.

  • Potential of scale-up optimization: protein yield can be increased by more than 5 to 10 times by Process Development. The Antagen’s expression system can easily reach the yield of 3 ~ 5 g/l protein, and greatly reduce production cost.

References:
  1. https://en.wikipedia.org/wiki/Protein_production#Cell-free_systems
  2. Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004 Nov;22(11):1393-8.
  3. https://en.wikipedia.org/wiki/Chinese_hamster_ovary_cell
  4. Xu et al. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol. 2011 Jul 31;29(8):735-41.
  5. Wurm FM, Hacker D. First CHO genome. Nat Biotechnol. 2011 Aug 5;29(8):718-20.
  6. Lewis NE et al. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nat Biotechnol. 2013 Aug;31(8):759-65.
  7. Xu N et al. Comparative Proteomic Analysis of Three Chinese Hamster Ovary (CHO) Host Cells. Biochem Eng J. 2017 Aug 15;124:122-129.
  8. https://en.wikipedia.org/wiki/HEK_293_cells
  9. https://www.atcc.org/Products/All/CRL-1573.aspx#characteristics
  10. https://www.atcc.org/Products/All/CRL-3216.aspx#characteristics
  11. Geisse S, Voedisch B. Transient expression technologies: past, present, and future. Methods Mol Biol. 2012;899:203-19.