Dialysis buffer for recombinant protein which has urea
Answers
Explanation:
The problem is solved differently for different proteins, depending on their physicochemical properties.
The major danger of abrupt changes in urea (or any other denaturing agent you may use) is protein aggregation. As a general case, one expects protein globules to form spontaneously as soon as the denaturant is removed. The trick is that often a protein needs more time and a certain path to acquire the native fold, whereas misfolded, inactive species are produced rapidly in a variety of possible folding paths. When you decrease urea stepwise you produce a kind of path for proper folding, giving the protein both time and the opportunity to go back to correct the fold. When you drop urea sharply aggregates are formed quickly and, most tragedically, irreversibly.
Remark that this situation is not universal! Some proteins (among them rhodanese) are most susceptible to aggregation, for example, at 4M urea. The best way to renature a protein is to be found empirically.
Answer:
AnsweR :
In a many cases and in several host systems, recombinant proteins accumulate intracellularly in insoluble aggregates. The proteins in these so-called inclusion bodies are mostly inactive and denatured. In addition, dimers and multimers may be present. However, the expression of recombinant proteins in inclusion bodies can also be advantageous:
- the recombinant protein deposited in inclusion bodies can be 50% or more of the total cellular protein.
- the inclusion bodies often contain almost exclusively the overexpressed protein.
- in inclusion bodies the protein is protected from proteolytic degradation.
- expression in inclusion bodies will protect the cell against the toxicity of the recombinant protein.