Hi All, I sincerely appreciate all of your invaluable feedback and suggestions.
Your insights have greatly enhanced my understanding, allowing me to make a well-informed decision regarding the selection of cells. Thank you for your support. I will keep you all posted as I move forward and may ask more questions. Regards, Anamika On Thu, 12 Feb 2026 at 04:33, David Briggs < [email protected]> wrote: > 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 | 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 | 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.* > > 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 > -- Anamika Singh, Ph.D. Post-Doctoral Scholar ######################################################################## 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/
