Sponsored by: Thermo Fisher Scientific
Session ends: April 17th, 2015, 3:00pm MST
Answers by: Jonathan Zmuda, Ph.D. , Thermo Fisher Scientific, Inc.
This Ask the Expert will address questions in regards to transient protein production in mammalian cells. Mammalian transient expression allows for the rapid generation, purification, and characterization of milligram to gram quantities of secreted or intracellular recombinant proteins for therapeutic, functional, and structural studies.
Questions may include:
- Getting started with Mammalian Transient Expression Systems
- Key Elements Necessary for the Establishment of a Mammalian Transient Production System
- Scaling Transient Protein Production to Accommodate a Wide Variety of Early Discovery Studies and Applications
- Optimizing the Transient Protein Production Process
- Tools and Strategies for Purification and Evaluation of Transiently Expressed Proteins
Who Should Attend?
Scientists interested in setting up a rapid and robust mammalian expression process to produce milligram to gram quantities of recombinant protein.
Best practices for maintaining your cells before, during and after transfection:
Cells used for transient protein expression should be well characterized in terms of their growth rates/doubling times, growth profiles to maximum viable cell density and passage stability over time. From the time of thaw, care should be taken to ensure that cells are routinely sub-cultured when cells reach approximately 1/3 to 1Ž2 of their maximal viable cell density; cells should not be over-split before they reach log phase growth, neither should they be allowed to grow to exceedingly high densities before sub-culturing. Viability at the time of transfection should be high (~95% or greater) and viability and viable cell density should be monitored post-transfection to understand these parameters in your system.
Ensuring optimal complexation of your DNA and transfection reagent:
All transfection reagents are different from one another, thus there is no one best method for optimal transfection complexation. During optimization, volumes of DNA and transfection reagents should be determined empirically, as well as your choice of complexation medium, complexation temperatures and times.
Please take advantage of the opportunity to ask our expert a question and participate in a lively discussion on transient protein production in mammalian cells! We will also be posting tips, so check in regularly.
Questions & Answers
Most often researchers will use simple ELISAs or other immunoassay formats to check for antibody expression. These methods require relatively commonplace equipment and reagents, but can take longer to get to results. For serum and protein free media, if your antibody titers are high, you can actually get a relatively accurate look at antibody concentrations […]» Read MoreThe first step is to identify a serum-free media formulation compatible with the cell line you are using. Typically serum is removed from cultures in a gradual, step-wise approach, replacing the serum containing media with the new serum free media with reduced levels of FBS in it. Wean the cells from the FBS by replacing […]» Read MoreThe use of DMSO to enhance protein titers has been widely reported in the literature, however, we do not employ this method due to the toxic nature of DMSO as well as the undefined nature of its effects on cells. For certain cell lines, it may be advantageous to reduce the temperature of the cultures […]» Read MoreAbsolutely. While insect cells are still used quite a bit for expressing intracellular proteins, HEK293 cells also do a good job of expressing non-secreted proteins.» Read MoreMany researchers routinely use HEK293 cells at 1L scales in 3L shake flasks and at even larger volumes in 10L or 20L Wave bags or benchtop bioreactors. Certainly CHO cells can also be scaled up, as the vast majority of biotherapuetics are made in CHO cells at multi thousand-liter scales. Scaling up transient systems from […]» Read MoreIt is likely not simply an issue of using a “richer” media, but more so a better (i.e. possibly different) media that allows for more rapid growth of your cells so that the highest percentage of cells as possible are actively dividing at the time of transfection to promote DNA entry into the nucleus. There […]» Read MoreUnfortunately, this is a very difficult question to answer, as every step and each procedure plays a role in the success of your transfection. Always start be ensuring that your cells are as healthy as possible and have been cultured under controlled conditions, not letting the cells density get too high, or be too low, […]» Read MoreCells used for transient protein expression should be meticulously maintained from the time of thaw throughout their useful lifespan. Deleterious cell culture conditions can negatively impact not just your next transfection, but all transfections from that point forward. Cells should be well-characterized in terms of their growth rates and log phase growth range and should […]» Read MoreHEK293 is typically the cell line of choice for mammalian transient transfection due to their ease of transfection, high levels of protein expression and robust workflows. When proteins are being expressed for the purposes of therapeutic development, CHO cells tend to be the cell line of choice, as approximately 70% of licensed biotherapeutic proteins are […]» Read More