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 On Wed, Jun 16, 2021 at 7:09 PM Chun Luo <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> *On Behalf Of *P. H > *Sent:* Wednesday, June 16, 2021 3:19 PM > *To:* 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 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/
