ABSTRACT
@#Naturally split Npu DnaE intein can mediate rapid trans-splicing and C-cleavage, which is of great use in many aspects of protein engineering. However, the degradation of NpuC during expression and purification reduces the yield and purity of recombinant protein. N2C, an extended NpuN2-containing N-terminal NpuC fragment, was constructed to improve NpuC stability. N2C was expressed in BL21(DE3) and purified by affinity chromatography. The degradation ratio was calculated by ImageJ, and the factors affecting the C-terminal cleavage reaction of intein, such as temperature, DTT concentration and N/C ratio, were also investigated. The results showed that N2C lowered the proportion of degradation to 2.7%-7.2% and the yield of C-terminal cleavage reached 90% in 30 min at 37 °C with an N/C ratio of 5∶1 catalyzed by 1 mmol/L DTT. N2C can not only improve the stability of NpuC in Escherichia coli expression system, but also retain the activity of C-terminal cleavage reaction, which is of great significance for its application in protein purification.
ABSTRACT
Npu DnaE intein was used to produce some large proteins,which were difficult to obtain through conventional expression systems.A T7 expression system was described,by which the gene of T7 RNA polymerase is split into two pieces,and each piece fuses with Npu DnaE N-and C-terminal sequences respectively.Functional T7 RNA polymerase is created by mixing the two kinds of fusion constructs in vitro.The approach of split intein-mediated production of large proteins,in theory,readily generalizable to the purification of other large,cytotoxic or membrane proteins.