QMUL Science Alive: Protein expression and purification

E. coli bacteria are a common host for the expression of recombinant proteins used in a wide range of applications. Plasmids are circular DNA constructs which may encode instructions for the synthesis of an engineered protein by the desired host cells. Following transformation and selection of bacterial colonies containing the plasmid, the recombinant protein is expressed and purified. Poly-histidine tagging and Nickel ion chromatography are commonly used to isolate large quantities of highly-purified target proteins. This video will demonstrate the procedures involved, including expression of a His-tagged recombinant protein in bacteria, purification of the protein using Nickel ion affinity chromatography, and analysis using SDS-PAGE. Protein expression and purification is presented by James Wright and directed by Rosemary Clyne.

[@calebsawe8307]

James Wright is a good teacher. It was almost I was there with him. I can picture the entire process. He also gave details and principles behind why certain reagents are used. Awesome video. Would like it 100 times if I could.

[@rolfw2336]

I appreciate the buffer compositions that you overlayed on the video.

[@Teragarm]

Great video! and for everyone alarmed by him not wearing gloves, it's very normal in bacteriology research!

[@robweishart]

Thank you for that great introduction and clear explanation!

[@naemibickmeier4013]

You just made my day. Thank you so much.

[@78rydwan]

Great job, wish you all the best with your research.

[@jannabee3314]

Took me 8 weeks to successfully express a protein, it's pretty simple in theory but doesn't always work smoothly in the lab, great video though!

[@intuitivealpha328]

how difficult are somatropes?

[@danjbro]

That's the truth!

[@ericwuluglayjr6060]

That is precisely what I was looking for, thanks once more.

[@titi14fan]

Thank you; it was brilliant

[@itsmemadle756]

Really thank you for the explanation of such a complicated process in a simpler way

[@jonbuitbuitrago6867]

Thanks for this lab work divulgation

[@all_about_microbiology_]

Very Useful Video , Thanks for this 👍

[@winter4ever567]

Great Video! James, where ever you are, I hope you're doing well. Cheers bud

[@nels6991]

Great Video Thank you!
Does anyone have any good sources for how this is done at the large industrial scale?

[@j.m.e.r8539]

Nice video and very well explication! Congrats!

[@AA-gl1dr]

thank you so much!

[@danielrodriguezaguilar3867]

Cómo me gustaría que mi laboratorio estuviera así de equipado u.u
Excelente vídeo, me encantó :3

[@markh1652]

thanks for sharing!

[@Drukonosz]

Nice material!

[@qamarhennawi9137]

Thank you so much

[@hesnayigit8840]

Thank you very much, this was a very good show and tell for the students.

[@AinaSakeenah]

Thank you

[@houtsereywatana7539]

thank you for explaining

[@afifahusna6561]

i clapped when i see a beautiful nice band u got after purification. nice video

[@SaraMonicaLeonUlloa]

Thank you for this video! It's cool how you do this in a lab!

[@musabiremaalexis4477]

Hello,Thank you for the video

[@ericwuluglayjr6060]

THANKS ALOT ❤

[@mkcd3829]

You’re the best

[@xima7140]

Hi, can we store the supernatant obtained after sonication and centrifugation that has expressed proteins?

[@SouravGhosh-pn7bl]

Do you also use any eukaryotic system for protein expression followed by purification than bacterial system?

[@nels6991]

Are these E. coli producing proteins by cytosolic production, Inclusion body formation, secretory production, or excretory production?

[@mlearningplus5183]

Could you please guide cloning techniques step by step

[@Artas1984]

That plasmid is missing a marker needed for individual colonies separation by color: a screening marker, that is in the MCS region.

[@nels6991]

the screen is already done by inserting the antibiotic resistance gene. A color marker would only be needed if the plasmids were a mix of plasmids with the target sequence and plasmids without the target sequence.

[@Tepmodify]

I like video. Interesting . good describe

[@Sulbee]

How do you talk so fast :D I was watching this for exam prep, and I had to slow it down :D Thanks for this though :)

[@rumjhumtyagi2880]

if the protein isn't expressed, what does that indicate? what mistakes could I have made?

[@Structuralbiology]

Inducer is not able to remove repressor or other reasons may be that your bacterial does not allow the expression of protein

[@drshinwarishinwari1253]

No gloves 😱

[@shilpawaduwawara9529]

Yea!! Freaks me out! Good video except for that though.

[@drshinwarishinwari1253]

Shilpa Waduwawara 😀

[@nicolasalarcon7162]

If you're working with microorganisms near to a flame. Its dangerous wear gloves. Also the flame sterilize very good the surroundings and usually the bacteria had a antibiotic resistence to keep it clean.

[@fatimahayssam120]

is it a cell biology lab or biochemistry?

[@jannabee3314]

It can be any sort of biological or chemistry lab, I did this exact same procedure in a Biotechnology lab which was part of a school of chemistry.

[@DrGhosh]

1) The optical density of the E.Coli culture during IPTG induction and IPTG concentration? 2)Too much Ni-beads for that volume of the supernatant, so probably there was nonspecific binding of other protein to the beads leads to the tiny bands in elution lane. 3)The safety issue while the use of glass beaker for sonication. 4) no sample loading for wash step as well as beads!!

[@anisnadyraahmadzifruddin6820]

where's your glove tho.........

[@alexlee6859]

So uh, what's he cooking?

[@ChompNom]

gloves?

[@richardfever]

Wubbywub the First thing that I noticed

[@xima7140]

Not needed

[@tremeramcbean3719]

Why aren’t you weren’t gloves?

[@adronung1892]

No gloves?

[@johnhuang113]

not feeling good without the gloves

[@danjbro]

Gloves dude...gloves!

[@runningcookiess]

It’s not safe to do experiments without gloves and goggles