I want to express a 37 a.a, 43 a.a and 55 a.a peptides using pET His6 Sumo TEV expression vector with BioBrick polypromoter restriction sites (14-S). the vector host is XL1 blue and the company dosn't provide any information about induction of that strain. it is low copy plasmid and the company provide it in as bacteria in agar stab. actually i find a very good vector from invitrogen "pET SUMO Expression System" but it is really expensive. do you have any suggestion? i dont have any experience in expression and cloning. any help is appreciated.
https://www.addgene.org/48313/
This is a pET vector variant having N terminal tags to help in your downstream protein purification.Try the following method.
After cloning your gene of interest into this plasmid, just transform it into chemically competent BL21 DE3 strain of E.Coli and grow it for an hour in absence of antibiotic stress. pET class of vectors express well in the DE3 variant of BL21 strain. [Check that you are working with BL21 DE3 and not just BL21 and that the cells have been rendered competent by chemical treatment]. Then plate on appropriate antibiotic resistance plates. Once you get colonies, pick and make glycerol cell stocks. Use these to carry out an expression test. Meanwhile in parallel, isolate plasmid from the corresponding batches and send for sequencing to confirm presence of correct gene. For medium scale expression test, just grow a pinch of your glycerol cell stock in 5ml of LB having appropriate antibiotic for 15 hours over the night. Next day, subculture by taking 1.5ml of the overnight culture and add to 250 ml of fresh LB containing appropriate antibiotic. Allow it to grow till OD at 600nm reaches around 0.6-0.8. Once there, just add 0.5 mM IPTG and grow it for either 4hours at 37 degree centigrade or 16 hours at 20 degree centigrade. Keep the shaker at 180-200.
Keep in mind that a lot of work needs to be done to standardise this protocol for your protein. One area is to vary your IPTG concentration as 0.5mM, 1mM, 1.5mM and see which works best. Also work with the time and temperature of growth as mentioned earlier.
Don't forget to keep collecting samples of your culture every hour after induction to determine at what time there is maximum over expression of your protein. Check that there is no leaky expression by also collecting samples of culture prior to induction.
Another aspect to watch out is if you don't get protein in the soluble fraction, then it might be going into inclusion body aggregates. Check for that as well.
It seems a lot but with time and practice it won't be confusing at all.
Best wishes.
Generally it is not easy to express small peptides below 10 kDa. Small peptides are often flexible and thus protease sensitive. The most common solution is to fuse it to expression partner which is then cleaved off after purification. Typically, an expression tag will provide better expression of small proteins.
SUMO is a wonderful tag to try - it generally increases expression levels and solubility. You can cleave the SUMO tag off in a wide range of conditions using the SUMO protease which leaves no cloning artefacts on your protein of interest.
MBP allows for easy purification and then can be cleaved off. Additionally, if this is a transmembrane peptide or something that may be toxic to cells an MBP or GST tag will enhance solubility and give higher expression.
If you need help with protein expression and purification,the following link may help you:
http://www.biologicscorp.com/protein-expression-purification/
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