Dear Kavya, I wanted to share with you that we have faced the same issue with a few projects. In addition to the great suggestions given earlier, I recommend trying out fusion proteins, such as the surface entropy reduction MBP mutant, or using protein binding partners like antibodies or natural binders. These strategies can help alter the protein properties and facilitate new crystal contacts. Additionally, it may be worth experimenting with reduced salt concentrations or using a salt-free buffer, akin to using water in place of a buffer.
PMID: 32541044 PMID: 26850170 Wishing you the best of luck, Tao-Hsin Chang > On Feb 5, 2024, at 10:05 AM, kavyashreem <kavyashr...@instem.res.in> wrote: > > Dear all, > > Thank you all for your valuable experiences, inputs and references!! I shall > try them and hope for some good news! Its good to know there are so many > examples of crystallization at such high concentrations. > > A curious question - is it logical to say that if the protein is highly > charged (either negative or positive), it is likely to be more soluble and > resist crystallization due to electrostatic repulsion? Our protein has highly > positively charged surface, although with some small negative patches. > > Following are some of the suggestions indicated: > > 1. Use water (will try) > > 2. Change buffers, pH temperature - (done) > > 3. Seeding (will try) > > 4. Methylating lysines (will try!!) > > 5. Surface entropy reduction (ongoing) > > 6. Optimize constructs (done) > > 7. Different ratios (done) > > 8. Keep concentrating (will try, the problem is yield is low!) > > 9. High salt concentrations (will try) > > 10. Organic solvents (though about it) > > 11. Mutations (ongoing) > > Thank you > > Regards > > Kavya > > On 2024-02-05 15:57, kavyashreem wrote: > >> Dear All, >> >> Has anyone worked on a protein which is highly soluble even at 80mg/ml? >> >> We have one such candidate, which does not precipitate even at 80mg/ml >> instead forms phase separated globules in crystallization plate, which >> eventually hardens over a period of 1 to 1.5 months (which is florescent >> under UV microscope.) >> >> We tried screening at different pH, but failed to get any hits. >> >> Since we got few conditions in which the phase separated globules >> solidified, we focused on them and expanded with 120mg/ml protein, still >> there were not visible precipitates except for the phase separation. This >> has been a challenging target so far. We have tried with different >> constructs, which unfortunately are not soluble! >> >> Does POMs help in such cases? Or do you have any other suggestion. >> >> Thank you >> >> Regards >> >> Kavya >> >> >> >> >> >> CONFIDENTIAL: This email and any files transmitted with it are confidential >> and intended solely for the use of the individual or group to whom they are >> addressed. If you have received this email in error, please notify the >> sender immediately and delete this e-mail from your system. It is NOT >> AUTHORISED to copy, forward, or in any way reveal the contents of this >> message to anyone. >> >> >> >> >> CONFIDENTIAL: This email and any files transmitted with it are confidential >> and intended solely for the use of the individual or group to whom they are >> addressed. If you have received this email in error, please notify the >> sender immediately and delete this e-mail from your system. It is NOT >> AUTHORISED to copy, forward, or in any way reveal the contents of this >> message to anyone. >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> >> -- >> >> >> ********* THIS IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM >> **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT >> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE >> FROM ADDRESS AND URLs********* >> > > > > > > CONFIDENTIAL: This email and any files transmitted with it are confidential > and intended solely for the use of the individual or group to whom they are > addressed. If you have received this email in error, please notify the sender > immediately and delete this e-mail from your system. It is NOT AUTHORISED to > copy, forward, or in any way reveal the contents of this message to anyone. > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> ######################################################################## 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/
