What is the size of protein u r purifying??

For ur questions,, here r the answers

1. One way u can push a protein into inclusion bodies is by inducing with high amounts of IPTG,, check how much IPTG u had added in both the cases..

2. Ideally prefer induction temperature between 18 to 240C. Only when u r 100 % sure if ur protein is insoluble then u can consider 370C for induction where u can save lot of time.

3. Yes ur protein will loose its all activity and structure in denaturing conditions. Retaining them would depend on ur refolding efficiency

4. I advice prefer 8M Urea for denaturing as GuHCl is not compatible with SDS gel. Refolding buffer constituents varies from proteins to proteins. Minimum common things are a buffer of ur choice, high NaCL, KCl, MgCl2 and u have to try ur luck by varying many other components like Glycerol, sucrose or PEG for stability in various proportions, like wise DTT or TCEP or BME for reducing conditions again try with different proportions.  U may need to try with or without Triton x 100 and 1M urea. If u r facing aggregation problem u can add up to 0.5 M l-Arginine to prevent aggregation.

All da best!!

Hi Kanti

In this paper: http://link.springer.com/article/10.1007%2Fs00253-012-4548-4

The insoluble fraction was isolated, denatured and refolded. See below.

The pellets were dissolved in 6 M guanidine and 50 mM Tris–Cl, pH 8. The recombinant protein was purified by affinity chromatography on Ni column (HiTrap, GE) preequilibrated with 6 M urea, 50 mM Tris–Cl (pH 8.0), 300 mM NaCl, and 5 mM imidazole. The fractions corresponding to the target protein were pooled and dialyzed overnight at 4 °C against the following buffer [6 M urea, 50 mM Tris–Cl (pH 8), and 300 mM NaCl] to remove imidazole. Afterwards, the sample (8 mL at 0.6 mgmL−1)

was reduced with 10 mM of β-mercaptoethanol for 1 h at 4 °C and diluted drop by drop into 500 mL of refolding buffer at pH 9.0 (50 mM 3-cyclo-hexylamino-ethylsulfonic

acid (CHES); 10 mM β-mercaptoethanol and 10 % glycerol) previously chilled.

Hi Kanti,

your problem - leaky expression – is quite common in case of pET vectors and BL21(DE3) cells. The simplest way to suppress it is to add glucose to culture media. As we know from biochemistry textbooks glucose is a suppressor of lac system :).

Addition of glucose should start at plating of transformed BL cells. Few hours before transformation we spread 0.2 ml sterile 50% glucose on the antibiotics containing plate, and use this for growing colonies. The starter culture should contain about 0.5-1% glucose as well. It is a good practice to prepare larger volume starter culture and divide it before induction so you can check several IPTG concentrations for induction. It looks like your protein has a tendency to form aggregates, so be careful. The success of your first experiment shows that it is not a very difficult case, but it is important to make fresh transformations for every expression. I bet that you picked colonies from the same plate for the second experiment when you got inclusion bodies.

In general it is better if you can have your protein in soluble form but many proteins can be refolded even if they were produced in form of inclusion bodies.