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 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/
