Hi there,

I assume that you are going to purify the protein eventually.

The methodology depends greatly on the design of the expressing construct and purification preferences. For example, if  expression level is high and the protein is tagged, I would start and isolate soluble fraction and do affinity purification (Ni or Flag) in the presence of low concentration of b-ME (1-2 mM). Then, without time break and avoiding freezing the eluted material, I would load the top eluted fractions onto the second column equilibrtaed in the buffer with high freshly added DTT (5 mM). The choice of that column depends on the protein properties and purity of the material obtained from the step one purification. It can be size-exclusion or ion-exchange chromatography, or both (sequentially), if needed. If the protein has some kind of activity that is easy to detect and monitor during purification, it should be done as it will tell your great deal about the quality of your prep. If expression levels are rather low or the amount  of the soluble protein is low, I would try and develop a more conventional protocol (using multiple column purification steps) that utilize buffers with relatively high concentration of DTT (5-10 mM) from the very beginning (lysis buffer and so on). 

Also, it might be not a bad idea to monitor formation of disulfide bonds in eluted fractions in SDS gels in the presence or absence of DTT (a portion of the eluted fraction should be buffer exchanged/dialyzed prior this analysis). Migration of the protein will change dependent on the DTT presence and that will tell you about functionality of the Cys groups in the purified protein.

Try to work fast and and use only cold reagents/buffers. Avoid, if possible, in-between-purification steps freezdowns and prolonged keeping the samples at +4C.

Hope that will help.

Good luck. 

Hi,

every sigle case is differs one from another. It is significant how many disulphide bridges there are in your protein of interest, a distance between cysteins and in what order the cysteins form bonds. I worked with proteins with 4 Cys-Cys bridges using E.coli BL21 (DE3) strain for production of recombinant fusion protein. Bridging of Cys occured during cell lysis step, when fusion protein released from high-reduction-potential cytoplasm of E.coli cells to lysis buffer. So, this step should be carried out at low temperature (0-4oC) because of minimum-energy state. Almost always (in my cases) proteins folded correctly spontaneously during lysis step. That should be proved eventually by CD-spectroscopy of purified target protein. Only twice (out of ~20-30 purifications of different 4-Cys-Cys proteins) I had problems with correct folding and had to resort to refolding. By the way, the proper strain, like Origami B, gave much worse results, that is why I retreated from it usage.

Hi there,

I assume that you are going to purify the protein eventually.

The methodology depends greatly on the design of the expressing construct and purification preferences. For example, if  expression level is high and the protein is tagged, I would start and isolate soluble fraction and do affinity purification (Ni or Flag) in the presence of low concentration of b-ME (1-2 mM). Then, without time break and avoiding freezing the eluted material, I would load the top eluted fractions onto the second column equilibrtaed in the buffer with high freshly added DTT (5 mM). The choice of that column depends on the protein properties and purity of the material obtained from the step one purification. It can be size-exclusion or ion-exchange chromatography, or both (sequentially), if needed. If the protein has some kind of activity that is easy to detect and monitor during purification, it should be done as it will tell your great deal about the quality of your prep. If expression levels are rather low or the amount  of the soluble protein is low, I would try and develop a more conventional protocol (using multiple column purification steps) that utilize buffers with relatively high concentration of DTT (5-10 mM) from the very beginning (lysis buffer and so on). 

Also, it might be not a bad idea to monitor formation of disulfide bonds in eluted fractions in SDS gels in the presence or absence of DTT (a portion of the eluted fraction should be buffer exchanged/dialyzed prior this analysis). Migration of the protein will change dependent on the DTT presence and that will tell you about functionality of the Cys groups in the purified protein.

Try to work fast and and use only cold reagents/buffers. Avoid, if possible, in-between-purification steps freezdowns and prolonged keeping the samples at +4C.

Hope that will help.

Good luck. 

Hello Clelton,

I suppose that you may have had a look at them, but just in case, here are reviews by a specialist of recombinant protein expression : de Marco, A.2009. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb. Cell Factories 8, 26, and de Marco, A. 2012. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb; Cell Factories 11,129

In a past I was working with 5 kDa protein with 6 cysteines. What helped me a lot was a fusion with a thioredoxin tag. Later improvement was achieved growing the cells in rich TB or autoinduction medium and inducing at 18 C ON

Article Screening of fusion partners for high yield expression and p...

As Juliius mentioned screen through vectors also the dsbA and dsbC plus coexpression strategies with them.Also check out possible problems with Cysteins not involved on disulphide bridges:See Trave papers mutating 'bad' cysteines out.Can be very important.Also test the respective strains described in the Novagen manual (Origami).Otherwise important to know more about the protein.See papers e.g. about the receptor RAGE.

Clelton A. Santos Have you tried Shuffle strain? It also depends how many disulfide bonds your protein has and the protein boundary of the recombinant protein. I have been working on purification of membrane protein and extracellular matrix proteins that have a range of S-S bonds, ranging from 2 to 17. Have you tried CyDisCo technology? I addition, You ca try auto induction medium/ 16 C temp with 0.01 mM IPTG concentration in minimal salt medium. Having said that I Should also mention that purification of soluble ,properly di-sulfide bonded proteins is very tough, uncertain but possible under defined condition

agree with@ Ivan V Bogdanov

regards