Hi Marco, Thank you for the reply and this information.
I liked the idea of using this plasmid for all three expression systems. We will check the virus titer before infecting insect cells for optimum protein expression. I have not tried the neomycin version of the plasmid, but I will, too. Thank you again for your time and help. Best wishes, Mohinder ------------------------------------------ "Whatever you’re meant to do, do it now. The conditions are always impossible.” Doris Lessing ----------------------------------------------------------------------------------------------------------- > On 1 Nov 2024, at 10:52, Mazzorana, Marco (DLSLtd,RAL,LSCI) > <[email protected]> wrote: > > Hi Mohinder, > I successfully used modified pTriEx vectors from the pOPIN series from > OPPF-UK (now PP-UK) to express a number of proteins with baculovirus as well > as HEK293T cells. > > These vectors are great and allow quick, easy, and modular cloning of the > same construct with different tags at the N- and/or C- termini, to best adapt > to your requirements. > > Should you wish to use them for bacterial expression, my suggestion is always > to go for rich (e.g. TB-based) autoinduction media and long expressions (8 > hour 37C followed by 64-70 hrs at 18C). With the baculo and human cells the > work is (as usual) trickier in finding the right ratios of virus or DNA for > the transfection. > Do also consider the neomycin resistant variants (pOPINneo vectors) for the > production of stable eukaryotic cell lines. > > The primary reference for them is: > https://academic.oup.com/nar/article/35/6/e45/1033077 > https://doi.org/10.1093/nar/gkm047 > > You can find most of these vectors from Addgene. Here are a few links: > - https://www.addgene.org/26042/ > - https://www.addgene.org/26045/ > - https://www.addgene.org/26044/ > - https://www.addgene.org/26043/ > - https://www.addgene.org/53536/ > - https://www.addgene.org/browse/article/6014/ > > Some of the protocols from the team of Ray Owens at the Rosalind Franklin > Institute are in their webpage > https://www.rfi.ac.uk/projects/protein-production-uk/ > > Hope this helps. > > Best wishes > > > Marco > > > > -- > Marco Mazzorana, PhD > MX beamline scientist > DR1-46, Diamond Light Source > Didcot (UK) > > Tel: +44 (0)1235 778643 > Email: [email protected] <mailto:[email protected]> > > > This e-mail and any attachments may contain confidential, copyright and or > privileged material, and are for the use of the intended addressee only. If > you are not the intended addressee or an authorised recipient of the > addressee please notify us of receipt by returning the e-mail and do not use, > copy, retain, distribute or disclose the information in or attached to the > e-mail. Any opinions expressed within this e-mail are those of the individual > and not necessarily of Diamond Light Source Ltd. > Diamond Light Source Ltd. cannot guarantee that this e-mail or any > attachments are free from viruses and we cannot accept liability for any > damage which you may sustain as a result of software viruses which may be > transmitted in or with the message. > Diamond Light Source Limited (company no. 4375679). Registered in England and > Wales with its registered office at Diamond House, Harwell Science and > Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom. > > To unsubscribe from the CCP4BB list, click the following link: > 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/
