Recombinant Protein Expression in E. coli: Part I

This classroom tutorial explains the fundamental concepts pertaining to recombinant protein expression in E. coli.Free for download, reuse and remix. CC-BY

[@rachealspruill9014]

Thank you so much for posting these recombinant protein expression videos. Very thorough and easy to understand explanations! As an undergrad in a new lab working on recombinant protein expression in BL21 DE3 E. coli, I find this extremely helpful in catching up to the learning curve. Very well done!!

[@KENNETHFRANCISRODRIGUES]

Thank you for your kind comments Racheal. Glad to be of service as an educator. All the best!

[@ArcticLifeWithAsad]

Thank you. To the point lecture

[@user-ug3su7ib9q]

Thank you so much!

[@rajghosh4485]

How can i insert gene of interest in the vector if it is assumed that specific restriction enzyme site isnt there. Can we use primers? Please elaborate

[@rameshbio9]

Great lecture....very simple and clear.... Thanks for posting

[@KENNETHFRANCISRODRIGUES]

Thank you Sir. Glad to be of service.

[@linaleal4920]

As for the insertion of a Hist Tag should it be after the protein's stop codon or before? If the transformation is done by double digestion and the restriction enzyme site is inside the MCS but not attached to the Hist Tag, will the tag bind to the protein? (assuming we are using a pET - 28a+ vector and EcoRI - HindIII restriction enzymes.

[@KENNETHFRANCISRODRIGUES]

Before STOP codon as it must be translated into 6X His. Thank you.

[@prachetaghosh2308]

thank you Sir .its totally clear now

[@KENNETHFRANCISRODRIGUES]

Thank you

[@himanipatel2681]

That was really helpful! Thanks. Also, I have a question. You mentioned something about introns. How exactly does introns affect the protein expression?

[@KENNETHFRANCISRODRIGUES]

Prokaryotic expression systems cannot splice out introns. If these are not removed the gene will not be in-frame and the protein will not be expresssed. Suggest cDNA or an intronless synthetic gene.

[@linaleal4920]

If the insert to the plasmid is cDNA, how does the T7 polymerase in E. coli translates it to protein? Shouldn't it be first transcribed and then translated?

[@KENNETHFRANCISRODRIGUES]

Thank you. Transcribed first, there is a ribosome binding site downstream of T7 which facilitates translation.

[@tushargupta8301]

Thank you so much for informative lecture sir. I had a question here where you told we would extract protien from ecoli by lysing ecoli. How will we isolate the protien from the mixture of lysed ecoli cells and protien..?

[@KENNETHFRANCISRODRIGUES]

Thank you, Sir. The E. coli can be lysed using sonication in a standard buffer. I use Phosphate Buffer. You have to sonicate the cells in buffer and then centrifuge the cells down. The supernatant will yield soluble protein and the pellet will contain inclusion bodies or membrane-bound proteins. You can then resolve this on a Poly Acrylamide Gel. More advanced laboratories use chromatography to separate the proteins. This can be done if your proteins are Histidine or GST tagged. All the best for your work. Stay safe.

[@tushargupta8301]

@@KENNETHFRANCISRODRIGUES thank you so much sir for the explanation...