I need to express new unknown phytase-like protein in some host to further study its properties (phytase activity, temp. tolerance etc), but have not much experience in protein expression work. I found paper by Huang (Env. Microbiol. 2011, 13(3), 747-757) in which authors made protein expression in E.coli BL21(DE3), but wonder if this strain would be good candidate because it contains phoA and AppA genes and in theory should produce own phytase which might affect the phytase expression level.
I would be extremely grateful if protein experts can advice me for or against using E.coli BL21(DE3) strain for phytase expression work and/ or recommend other expression strain (E.coli?) which could be used.
Hello German,
First, I would ask do you know that your novel phytase has some distinguishing characteristic (most straightforward would be MW) among the others native to BL21DE3? Or perhaps an antibody that is specific for your phytase and not the others?
Assuming there are discernible differences, I would definitely recommend BL21DE3. I use it for all of my protein expression and it works very well. Small-scale expression tests can be done very easily (especially if you have a reagent like B-PER https://www.thermofisher.com/order/catalog/product/78248). If expression in the soluble portion is poor, try lowering induction temperatures and extending the growth time (12-18hrs).
Unless the protein is cytotoxic, it's unlikely having other phytases will drastically change expression, as you'll be using a constitutive expression system induced by IPTG. The protein I express (and yield ~100mgs from 1L of BL21DE3) has a native E. coli version as well, and it doesn't affect anything.
Good luck!
Hi Kyle,
Thank you very much for quick, informative answer and hints!
Yes, I know exact nucleotide sequence of the protein, so guess could estimate MW. No other information is available - only that it resemble cysteine phytase - and nothing about if it is cytotoxic.
What about BL21(DE3)pLysS variant? Would it be better than just BL21(DE3)?
Thanks again!
German
Герман, добрый день.
Мне кажется, что можно предварительно проверить, есть ли фитазная активность у хозяйского штамма. И даже если она есть, навесьте на ваш белок гистидиновый хвост и очистите на колонке. В штамме BL21DE3 рекомбинантный белок может выпадать в тельца включения, так что придется подбирать условия для экспрессии. Но чтобы убедится, что вы отклонировали действительно фитазу, вам достаточно будет небольшого количества растворенного белка, а оно там всегда будет, даже если большая часть выпадет. Есть еще штамм Arctik Express, его можно попробовать, если нужно будет оптимизировать экспрессию с целью получения большего кол-ва растворимого белка.
German,
I would highly suggest checking the predicted MWs of the other phytases (e.g. cysteine phytase) and seeing how similar it is to your predicted phytase (I recommend ExPasy http://web.expasy.org/compute_pi/). If they are dissimilar you don't really need to mess with the pLysS variant - as in my experience it can cause more confusion than it's worth. However, if they are similar in MW it's worth a shot as if it is successful the pLysS variant will reduce the levels of native proteins.
I am weary of the pLysS strategy as any time you're actively promoting expression of a generic protease like Lysozyme you're going to get reduced efficiency of each cell and probably some off-target cleaving of your target. In some cases it can be very beneficial, though. So in summary, I would try regular Bl21DE3 first, then the pLysS variant if you have problems separating native from target (even after tagging, etc).
Hope this helps.
Спасибо, Оксана)
Поскольку я пока только планирую, могу менять стратегию как угодно -
Да, идея проверить фитазную активность конечно была - просто надо было определиться какой штамм лучше покупать И вообще можно ли использовать BL21DE3. Но похоже в любом случае можно начать с него.
Еще вопрос - в некоторых статьях люди клонируют без сигнального пептида и стоп кодона ТАА (например в pET-22b(+)).
Так лучше и сделать? Вообще используют разные векторы - как минимум я нашел pET32a(+), pET22b(+), pT23b(+), pET-20b(+),
pET19b. Есть ли принципиальная разница? Пока я остановился на pET-22b(+) - у него есть как раз His-Tag.
С приветом из снежного Хельсинки)
Hi Kyle,
Thanks again for your opinion about pLysS!
Seems it is better to try without it, at least at the beginning. And yes I am going to use
pET-22b(+) - it contains His-Tag.
German
BL21(DE3) cells are always the first cell type you should try for bacterial protein expression.
As long as your version contains a his-tag, or another tag, it will be easily separated from the endogenous phytase.
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