FWIW, many of the beta-lactamases fall into the same category: single step purification on an affinity resin, a few litres can give you 100 mg quantities, great spectrophotometric assay, several good substrates and many inhibitors, both covalent (teaching kinetics) and non-covalent.
brian On Wed, Jun 16, 2021 at 6:23 PM Peat, Tom (Manufacturing, Parkville) <tom.p...@csiro.au> wrote: > 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 > > 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 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > -- Brian Shoichet, Professor, UCSF latest science from the lab: *http://www.bkslab.org/ <http://www.bkslab.org/contact.php>* ######################################################################## 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/
