Application of DTT |CAS:3483-12-3|in the Reduction of Disulfide Bonds in Proteins

DTT is a small molecule organic reducing agent. DTT is mainly used in nucleic acid extraction/protection, IVD kits, reduction of disulfide bonds in proteins, electroless nickel plating, etc. This week, editors of YACOO introduce the application of DTT in the reduction of disulfide bonds in proteins.

In recent years, the incidence of diabetes has shown an upward trend year by year, and a large number of clinical practices have confirmed that the treatment of diabetes with recombinant human insulin injection can achieve ideal results.

At present, the renaturation method of recombinant human proinsulin fusion protein has the following technical defects:

(1) Dilution and renaturation: The increased volume is large, the dilution rate of the denaturant is too fast, and it is difficult to control, and the protein content during renaturation is low, generally below 2 mg/mL, and the renaturation rate is about 60%.

(2) Dialysis renaturation: It is not suitable for large-scale production, cannot be applied to the production scale, and is easy to form inactive proteins.

(3) On-column renaturation: the production process is complicated and the production cost is increased.

Therefore, it is of great significance to develop a renaturation method for recombinant human proinsulin fusion protein suitable for industrial production to overcome the defects of the technology.

CN105732820B provides a renaturation method for recombinant human proinsulin fusion protein, which specifically comprises the following steps:

1) fragment the Escherichia coli expressing the recombinant human proinsulin fusion protein, and collect inclusion bodies;

2) Wash the inclusion bodies with the inclusion body washing solution;

3) After removing the washing solution, add a denaturant to the inclusion bodies;

4) Add a final concentration of 10mM sulfhydryl reducing agent (DTT) to obtain a reduced protein solution;

5) Slowly add the prepared renaturation buffer to the reduced protein solution in step 4, and stir gently to renature the recombinant human proinsulin fusion protein to obtain an optimized renatured protein solution.

Compared with the prior technology, the present invention is directed to a recombinant human proinsulin fusion protein, the renaturation concentration is increased from 0.3-2 mg/mL in the prior technology to 5-10 mg/mL, and the renaturation rate is increased to 80%-90% , the subsequent process can be simply purified. The reagents used in this patent are simple and easy to obtain, and the operation of each step is simple, fast and efficient, and all the operation processes are carried out at room temperature or 4°C, which greatly reduces the production cost and is suitable for industrialized large-scale production of recombinant human insulin.

References

CN105732820B A kind of renaturation method of recombinant human proinsulin fusion protein