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

[rachealspruill]

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!!

[rameshbio]

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

[tushargupta]

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..?

[linaleal]

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?

[linaleal]

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.

[rajghosh]

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

[himanipatel]

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