I agree with Roger that human CAII is easy to express in E. coli at high level and does not need a tag for purification- and that it is a good lesson for students to purify proteins without tags (they sometimes have better activity and crystallise better without having the tag). It is also easy to find things in a catalogue for binding studies (many small molecules with a sulfonamide moiety will bind) and assays are relatively straightforward to perform. Good recommendation! cheers, tom
Tom Peat, PhD Proteins Group Biomedical Program, CSIRO 343 Royal Parade Parkville, VIC, 3052 +613 9662 7304 +614 57 539 419 tom.p...@csiro.au ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Roger Rowlett <rrowl...@colgate.edu> Sent: Thursday, June 17, 2021 10:45 AM To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Looking for proteins for undergraduate biochemistry lab Human carbonic anhdydrase II is very expressible in E. coli, and purifiable in one step via affinity chromatography with para-aminobenzenesulfonamide affinity resin (which is relatively easy to make, and reusable for many years.) It can be assayed by stopped-flow spectrophotometry for CO2 hydration, by a timed colorimetric assay, or you can investigate it's esterase activity with p-nitrophenylacetate. This enzyme, as well as most other carbonic anhdydrases, is also easy to purify by a classical combination of anion exchange (Q-sepharose), hydrophobic interaction (butylsepharose), and gel exclusion polishing. The latter would be a good exercise for students in general protein purification optimization, which is an increasingly lost art. (Just had a conversation with one of the protein chemists ast BioGen who pretty much observed the same thing.) We routinely did classical purifications on tagless overexpressed proteins for crystallography work. The time saved in His-Tag purification is sometimes lost in cleaving the tag to make tagless protein for crystallography. A paper describing the purification procedure can be found in J. Chem. Ed. (https://doi.org/10.1021/ed075p1021) for the similar bovine carbonic anhydrase. An fun long-term undergraduate research training project might involve improving the esterase activity through student-initiated point mutations. I did this kind of parallel protein mutation project with my students in a biochemistry research training studio course I taught, often with one of my research target proteins. Teaching lab students can do all sorts of crazy things you might never prioritize in your funded research. Some of these crazy things turn out to be fun and interesting. One of my students insisted on making a mutation in a protein that seemed to have low chances of leading to a successful publication based on prior work. Lo and behold, that mutation turned out to be gold, and he was published within the year. I still can't believe it not only worked, but crystallized easily from the first screen and optimization for structure determination. Go figure. _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor, Emeritus Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu> On Wed, Jun 16, 2021 at 7:09 PM Chun Luo <c...@accelagen.com<mailto:c...@accelagen.com>> wrote: Many phosphatases, such as lambda phosphatase, have good soluble expression in E. coli. Their activity can be shown by simply colorimetric assay. From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> On Behalf Of P. H Sent: Wednesday, June 16, 2021 3:19 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Looking for proteins for undergraduate biochemistry lab Hello All, We are looking for some candidate proteins for an undergraduate level advanced biochemistry lab. They should be expressed in bacteria, simple enough to purify and it will be nice to perform some simple characterization experiments(binding assays, enzymatic assays). Any suggestions? Thank you in advance. Prerna gupta ________________________________ 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 ________________________________ 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/
