![]() Stable gene expression from a recombinant cell line has several advantages over transient transfection, including lower demand of plasmid DNA and transfection reagent, higher batch-to-batch consistency and easier scale-up. 2009 Gutiérrez-Granados et al., 2016 Bleckmann et al. However, it has several limitations when it comes to scale-up ( e.g., need for high DNA amounts, mixing of DNA with the transfection reagent, partial-to-complete medium replacement prior to transfection), commonly leading to increased manufacturing costs and batch-to-batch variability ( Ansorge et al. Transient transfection is a promising approach to produce a wide variety of recombinant proteins in short periods, suitable for target screening and at early stages of the development of biopharmaceutical products ( Puente-Massaguer et al. Virus-free expression in insect cells has been increasingly explored to circumvent BEVS-related drawbacks. Degradation of produced recombinant proteins is also likely to occur due to the release of intracellular proteases from the virus-lysed host cells, thus reducing recovery of the overexpressed protein ( Summers 1991 Farrell et al. Moreover, reports indicate that membrane and secreted proteins are frequently expressed poorly and heterogeneously ( McCarroll and King 1997), suggesting that the insect cell secretory pathway may be compromised due to the virus infection process ( Ailor and Betenbaugh 1999). Nevertheless, generating and preserving high-quality recombinant baculovirus is rather time-consuming and labor-intensive. It is used to produce the approved commercial vaccine FluBlok for human use ( Krammer and Palese 2015). The BEVS is flexible, rapid and often generates high titers of complex and multi-subunit gene products during the late phase of viral infection, even from the most challenging cellular locations (such as cell membranes, among others) ( Drugmand et al. When considering insect cells for the production of recombinant proteins to meet pharmaceutical needs ( Cox 2012 Krammer and Palese 2015), or to feed structural, functional and drug screening studies, the leading choice is the Baculovirus Expression Vector System (BEVS) ( Assenberg et al. The superiority of insect cells over bacteria or yeast for expressionof complex proteins is well-demonstrated ( Stolt-Bergner et al. Stable expression, insect Hi5 cells, RMCE, FACS, influenza VLPs Introduction Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development. Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein ( i.e., iCherry, as an intracellular protein model) or two proteins ( i.e., influenza HA and M1 for virus-like particles production, as an extracellular protein model). The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). ![]() In this study, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. Insect Trichoplusia ni High Five ™ (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |