I have read some papers concerning expression levels of proteins and instances of misfolding to due to "back up" during cellular processing.
I am expressing viral surface proteins and derived constructs in vitro in both 293F and Expicho. It is quite often that we will design a new construct and after purifcation, see that there is quite significant aggregation, and/or deleterious bands on the SDS-page. We often attribute this to a poor design. How likely is it that the protein is being overexpressed, causing the misfolding and aggregation? We typically add 900ug plasmid DNA per liter of transfection for 293F, and 1000ug per liter of Expicho.
Hi there,
The effect of the overproduction on the protein folding is very dependent on the protein itself: there are proteins which are able to fold properly and those which are prone to agregation mainly because the rate of synthesis is faster than the rate of folding. So what you may look for is to slow down translation and/or improve the quality of the folding (addition of chaperones or addition of additives known for lowering the process of aggregation). As a starter you may find some info in the following article:
Article Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses
Hi there,
The effect of the overproduction on the protein folding is very dependent on the protein itself: there are proteins which are able to fold properly and those which are prone to agregation mainly because the rate of synthesis is faster than the rate of folding. So what you may look for is to slow down translation and/or improve the quality of the folding (addition of chaperones or addition of additives known for lowering the process of aggregation). As a starter you may find some info in the following article:
Article Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses
I believe cellular homeostasis provides for production of chaperones that regulate production and folding of proteins, especially in bacteria.
Hi, Colin Mann.
My first question in order to answer yours is:
Are you having overexpression? How many mg or grams are you yielding with your production?
If you're having a overexpression, then, yes, maybe that's your problem ... misfolding is a very often reached problem in protein production and purification by molecular biology tech.
Some proteins may aggregate because they work as polymers and then, check if it is the case of the protein you're producing!
Hope it helps.
It depends on the protein of interest (foreign protein expressed in bacteria or mammalian cells) and really how much protein you are producing, and in what cell line and under what conditions,. Certainly over-expression can affect protein quality including post translational modifications such as N/O linked glycosylations which may be necessary for integrity/activity/folding/immunogenicity of protein. Over-expression is a very broad point, at lab scale we get low-high mg's/L, Industry grms/L.
Adriano Costa de Alcantara Ian R Wilkinson Expression for our different viral proteins is variable, especially based on each one of our egineered constructs. As a first check analysis, I purify these surface glycoproteins with fused 6xHiS with Ni-NTA columns. As a second check I use conformationally dependent antibody affinity columns. However, often if constructs do not show promise in the first check, they do not progress to the second.
So my worry is that some from the Nickel columns are well-designed constructs, but may be impacted by the level of cellular expression. For His-tag purifications, I typically get anywhere from 10 mg/Liter (Fcell)/200mg/L (CHO cell) to 100mg/L (Fcell)/2g/L (CHO cell). As you and others have said, each protein is different, but I am unaware of the predisposition of these proteins to aggregate during overexpression. I know from a protein expression standpoint, these engineered glycoproteins are typically more difficult to express properly, as they are typically trimerized membrane proteins which have been truncated to be soluble, and are decent sized.
In general some of our other viral proteins have no problem at around 100mg/L, and have very pure products, but I was just curious about the frequency and commonality of this issue.
Thank you for the information!
Hi, Colin Mann.
As you probably know and we all agree, it's very common to get aggregates when producing recombinant proteins. So, it is quite common too to get different results with different tags (His tag, antibody affinity, etc). So, usually, the best option is to try your protein of interest with a bunch of different tags and chaperones. It will bring cost to a higher degree, of course ... but often gives you best results faster. Is it possible for you? What do you think?
Hope it helps you!
In you description you state that "After Purification" you see aggregation? Do you not see this prior to purification?
If this is true then I have a few comments
1. Could well be a problem with buffer formulation. This has huge impacts on protein stability and can take a while to sort through. If pH is too close to pI point then this may well cause problems, but there are a number of other possible causes, buffer agent itself, salt (ionic strength) etc. There are numerous additives that can be added to aid stability.
2. Is SDS-PAGE done under reducing and non-reducing conditions. Heating in SDS PAGE may trigger aggregation/degradation especially at >90C. We use 65C for 15 minutes for some proteins.
3. Try using a different tag such as MBP which may aid in solubility and stability. Is the protein aggregated prior to IMAC column or is it just post elution? If so change the the conditions of the experiment for example elute with pH, EDTA or Histidine. Batch bind protein as this avoids over concentrating protein at the top of the column which may well drive aggregation.
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