Following up on Tom's comment with "that one weird trick that extracellular crystallographers don't want you to know*"
Let's assume you're working on a human protein. Make a big old sequence alignment of that protein from at least 10-20 different mammalian species. Looks at conservation of the N linked glycan sequons (N-X-S/T, where X≠Pro). Conserved = probably needed for fold or function - keep it in. Not conserved = probably just decoration. Get rid of it, either N>Q or the most common homologue residue (if human has an N but every other species you look at has an S at that position, go with S). This reduces complexity, makes your life easier and in my hands generally gives a well-behaved protein with comparable (sometimes better) expression than the wild type sequence. Combine this with GntI- cells or kifunensine ± Endoglycosidase H** treatment and you're probably close to the minimal viable glycosylated protein. Note when using kifunensine: pre-treat your cells for 24hr ahead of transfection, and keep it in the media throughout your expression. HTH, Dave *caveat emptor, of course. **Pngase F is commonly used, but - is sometimes sterically blocked from some glycan sites, and so doesn't give efficient or complete cleavage in native conditions. Also remember that Pngase actually cleaves the bond between CG and ND2, and replaces the ND2 with an oxygen, so it turns your Ns into Ds. -- Dr David C. Briggs CSci MRSB (he/him) Principal Laboratory Research Scientist, and Co-chair, Crick Staff Consultative forum Signalling and Structural Biology Lab 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=DRKG5wAAAAJ> == "Would it not be better if one could really 'see' whether molecules...were just as experiments suggested?" – Dorothy Hodgkin ________________________________ From: Tom Peat <[email protected]> Sent: Thursday, February 12, 2026 8:43:46 AM To: [email protected] <[email protected]>; David Briggs <[email protected]> Subject: Re: [ccp4bb] Guidance Needed for Secreted Protein Expression External Sender: Use caution. Hello Anamika, To add to what others have said- if you are producing the protein for crystallographic work, you may want to consider the question of glycosylation. There are specialised strains (e.g Lec8) that reduce the amount of glycosylation on the protein being produced. One can also add a specific inhibitor (e.g. kifunensine) to the cells to limit the amount of glycosylation. Best of luck, Tom ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of David Briggs <[email protected]> Sent: Thursday, February 12, 2026 7:27 PM To: [email protected] <[email protected]> Subject: Re: [ccp4bb] Guidance Needed for Secreted Protein Expression You don't often get email from [email protected]. Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> 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 email and any files transmitted with it may contain confidential information. If you believe you have received this email or any of its contents in error, please notify me immediately by return email and destroy this email. Do not use, disseminate, forward, print or copy any contents of an email received in error. 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