I am having trouble expressing a protein and have heard that co-transforming it can improve stability and increase expression, so I am looking to try this experiment. I only need the kinase domain from the original kinase, but it is expressed in low amounts, so I am trying to do this. I'm using a HIS6 tag and plan to utilize the TEV cleavage site to take the tag off afterwards.
If I were to design a construct for this experiment, could it be organized as follows?
pet28a: HIS6 tag and TEV cleavage site at N-Terminal + Kinase domain (1-200)
pet21b: not any tag at N-Terminal + C-terminal domain(201-500)
After this design, can I put both vectors into BL21(DE3)?
Would this result in expression with amino acids from 1-500 with HIS 6 tag and TEV site at N-terminal?
When designing like this, I was wondering if I should also put a linker or something. I'm completely new to this, so any recommendations for papers to read or sites to refer to would be appreciated.
I apologize if my question is too long.
Some proteins are expressed only in tiny amounts, depends on the protein. Potentially fusing them to some well expressed proteins like GST, GFP or MBP increases expression rate. Lower levels of induction at 20°C for 24h may increase yield. If you utilize larger fusion proteins which you intend to cleave via protease, make sure to include a linker sequence so the protease has access to the cleavage site. Something small like a His-Tag doesn't require a linker sequence.
If you want to express two proteins synchronously in comparable amounts, you may utilize either fusion expression or polycistronic translation. In case of the latter, between promoter and terminator you have multiple TATA-boxes that start the expression of different proteins from the same piece of mRNA, leading to somewhat comparable expression rates. In case of the former, you express one major polyprotein that gets cleaved into the single functional proteins in vivo by a protease, hence being quite stoichometrically accurate. This is how many viruses operate. The corresponding protease either has to be part of this polyprotein, or has to be encoded on the same/helper plasmid. Of course you could also cleave it after extraction in vitro. If using a helper plasmid, remember to use different selection markers so you can select for the presence of both (as it is the case with your examples of the two pET-vectors).
If your two domains don't assemble themselves via affinity into a functional split protein, you can still try linking them via spytag/spycatcher or via intein sequences which will "splice" the proteins into one, if the sites are accessible.
Dear Hyunjin Kim
is it a human kinase? Which specific domain you are interested?
i'm not expert on kinases but it is quite common that in E.coli you can observe low expression of proteins from eucariotic organisms.
in this case in general as Andre Marschall suggest the addiction of N terminal tags as GST, MBP, TRX, GB1. NusA can improve the expression level as well as the codon optimization of the Dna codifing the orf since in general the eucariotic present a lot of rare codons that can slow down the protein expression.
good luck
Manuele
Dear Hyunjin Kim
at the 7' 30'' on the following video
https://www.blogger.com/video.g?token=AD6v5dzPeA9ws3hcPvrO8fBABN8iFQ0dwI7GIHys9p8dwVtov4dGrKXH9PF91d9bVMYcLxTYY3XJesKt62Y-LsZcaHDCJKZbNW-DLUm_4p5-75Dbj1VZlKTlyyl2aQzf-EPwO3Ac6eBG
purbblished on my blog, proteoCool, you can find a list of N-terminal fusion tag that can improve the protein expression level.
best
Manuele
Some kinases aggregate due to phosphorylation. You can try to co-express the kinase domain with a phosphatase. Typically, Ser/Thr kinases are co-expressed with a Lambda phosphatase, and Tyr kinases with YopH.
If your goal is to increase protein expression, you might consider changing the His-tag to MBP, SUMO, or NUSA, or using a different host, such as mammalian cell lines or the SF9 insect model
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