Dear Colleagues, Two years ago, I solicited your help to identify candidate proteins for our protein ladder project. Thank you for all your suggestions, particularly to Erik Klontz for his tip to use the interleukin IL-1B.
Undergrads in my lab and I have constructed plasmids that express 10, 15, 20, 30, 40, 50, 60, 80, 100 kDa proteins at very high levels (50 ml of E. coli is sufficient for 20,000 lanes of each protein). The proteins are easy to purify via HIS tags, and the engineered immunoglobulin binding domains enable detection in Western blots. We also provide polycistronic expression plasmids to coexpress the 10, 30, 50, 100 kDa proteins on one plasmid and the 20, 40, 60, 80, 100 kDa proteins on a second plasmid. One can produce protein ladders for less than a penny a lane, vs about US$1 for many commercial ladders. We also expressed 150 and 250 kDa proteins but the expression levels are significantly less than for the others (no surprise). All the plasmids are available from Addgene. The plasmids could also be useful for teaching labs that might want robust expression using minimal equipment (the proteins can be expressed at room temperature). The manuscript describing the Penn State Protein Ladder system is now published at Scientific Reports: https://www.nature.com/articles/s41598-021-96051-x The Supplementary Information to the manuscript includes detailed instructions for expressing and purifying the ladder proteins. The Penn State press release can be viewed at: https://news.psu.edu/story/666355/2021/08/20/research/penn-state-protein-ladder-system And the link to the Addgene plasmids: https://www.addgene.org/browse/article/28219996/ Please feel free to tell your friends and colleagues about this resource. Postings on social media are welcome. Regards, Song --------------------------------------- Dr. Song Tan Verne M. Willaman Professor of Molecular Biology Director, Center for Eukaryotic Gene Regulation Dept of Biochemistry & Molecular Biology 468A North Frear Lab Penn State University University Park, PA 16802 email: [email protected] phone: 814-865-3355 web: http://www.personal.psu.edu/sxt30 > On Jan 6, 2019, at 6:14 PM, Tan, Song <[email protected]> wrote: > > Dear Colleagues, > > As a followup to my lab’s plasmids for making DNA molecular weight markers > for about one cent per lane (Henrici et al, Scientific Reports, 7:2438, 2017; > plasmids are available from Addgene), we are working on a similar project to > produce protein MW markers in E. coli. We are having problems finding 10-20 > kDa proteins that express solubly at high levels in E. coli and that migrate > appropriately on SDS-PAGE. We have eliminated multiple proteins including > thioredoxin and DHFR that express very well but that migrate anomalously > slowly on SDS-PAGE. > > So we are looking for proteins which fulfill the following criteria: > > 1. MW = 8-10 kDa or 18-20 kDa, > 2. Express as soluble protein in E. coli, > 3. Can be purified from E. coli with yields of greater than 10 mg/L culture, > 3. Migrate appropriately on SDS-PAGE. > > We hope to create a simple and inexpensive system for producing protein MW > ladders, and to distribute the plasmids through Addgene. Plan is to > coexpress multiple proteins using our polycistronic expression system to make > the production process simpler. > > If you know of proteins that fulfill all of the above criteria, please do > drop me a line. > > Regards and thanks, > > Song > > --------------------------------------- > > Dr. Song Tan > Professor of Biochemistry & Molecular Biology > Center for Eukaryotic Gene Regulation > Dept of Biochemistry & Molecular Biology > 108 Althouse Laboratory (office in 468A North Frear Lab) > Penn State University > University Park, PA 16802 > email: [email protected] > phone: 814-865-3355 fax: 814-863-7024 > web: http://www.personal.psu.edu/sxt30 ######################################################################## 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/
