Hi Anamika, I just want to echo much of what Kevin said:
Please don't think you are locked into specific media and cell types offered by a particular company, or their (normally outrageously expensive) proprietary transfection mixes. Good old fashion PEI-based transfection can still allow you to crank out mgs of your favourite protein, if you think about things carefully. Also, we routinely use Expi293 cells in Freestyle293 media. Expi media is 3x the cost of Freestyle media, but we rarely see 3x the yield. So why bother? In addition to your choice of Signal Peptide (there are some good papers talking about optimisation of SP for recombinant protein production) you want to think about whether you want a stable cell line (good for your wild-type "parent" construct, but can be slow to generate initially) or transients (allow you to be quick on your feet and move rapidly from mutant construct > protein, but you'll spend more money on plasmid prep kits). When screening SPs, I normally trial the native SP (it might be there for a reason!) alongside favorites like BM40 and IL2. You can also use adherent HEK293s (strongly recommend expanding up to gas-permeable hyperflasks for the final expression run) for semi-stable transfection. Reagents and media are cheaper and you don't require a shaking CO2 incubator, but time from construct > protein is typically 6-8 weeks, not days. Good luck! Dave -- Dr David C. Briggs CSci MRSB (he/him) Principal Laboratory Research Scientist (Signalling and Structural Biology Lab) and Co-chair, Crick Staff Consultative Forum The Francis Crick Institute London, UK Working hours: Mon-Fri 0900-1700 == about.me/david_briggs<https://about.me/david_briggs> | OrcID<https://orcid.org/0000-0002-9793-7339> | Google Scholar <https://scholar.google.co.uk/citations?user=DRKG5KwAAAAJ> == "Would it not be better if one could really 'see' whether molecules...were just as experiments suggested?" – Dorothy Hodgkin ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of Anamika Singh <[email protected]> Sent: 11 February 2026 15:07 To: [email protected] <[email protected]> Subject: [ccp4bb] Guidance Needed for Secreted Protein Expression External Sender: Use caution. Hello Everyone, I am seeking advice on selecting the appropriate cell lines for the expression of a secreted protein that is over 180 kDa in size. I am particularly interested in conducting expression experiments using mammalian cells. I have come across several research papers that mention the use of HEK-derived cell lines, such as HEK293 GnTI-, Expi293, and FreeStyle 293F. I would greatly appreciate your insights on how to choose the best cell line to achieve a high protein expression yield, as this specific protein has shown suboptimal expression in other systems. Thank you for your help. I look forward to your response. Regards, -- Anamika Singh, Ph.D. Research Associate ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
