Lecture 9/10 Applied Molecular Biology How do we make protein to purify? · Choose organism · Clone DNA is a suitable expression vector · Work our growth/induction protocol (each organism will express different proteins differently) particularly important if you want to express something that is toxic to the cells · Cell harvest and lysis methods Organisms · E.coli o High protein yields o Simple to clone and culture o No post-translational modification - e.g. wont work for a protein that needs glycosylating · Yeast o High cell yields o Relatively easy to culture o Some post-translational modification (some even hyper glycosylate, more than we want) · Eukaryotic cell lines (for more specific post translational modification) o Much harder - to transfect, prone to infection o Low yield o Exactly the right post-translational modification o Insect and mammalian cell lines used o Only used if you have no choice as also expensive Each gene must be surrounded by a start and stop codon, promotor and terminator and a ribosom binding site Vector-e.coli · Standard o Need inducible promoter, RBS and terminator o Toxicity considerations (if protein is toxic to cells) - affects choice of promoter. You need a promoter that can be controlled tightly (switched on or off) · Fusion Promoter RBS Fusion domain (optional) Gene o As above + fusion domain o Choose fusion domain according to purification strategy and nature of recombinant protein Terminator o Gene must be cloned in-frame with the fusion domain (insert must also be divisible by 3) o Fusion domain can be at either C or N terminal of encoded protein
Growth and induction - starting point · Choose strain of E.coli carefully . Must use freshly transformed cells otherwise yield will be poor · Culture single colony overnight in small volume (don't want a mix of cells) . Use this culture to inoculate, fresh, warm and large culture at ratio of 10:1 e.g. 100ml of overnight culture to 1l of broth · Grow to exponential stage and then induce expression · Induce expression via IPTG or change in temp etc · Incubate at 37 c for 3h and look at yield Growth and induction - refinement · Once you've chosen strain · Determine induction point (log phase, early lag, stationary etc.) · Determine best growth temp post induction to maximise yield (sometime lowing temp is better. Will cause production to slow down but then protein can be folded properly. Too fast and inclusion bodies formed) · Determine culture time to maximise yield e.g. lower temp means longer time · Set up lots of small cultures with many variations on induction point, growth temp and time. All needed to optimise expression of some proteins To identify which conditions are best, take 1ml of culture out of culture at various time points, temps etc., spin cells down and remove culture medium, take up in protein buffer and put in boiling water bath to lyse cells (will denature proteins but running on PAGE so ok) and run