Cloning, Expression, Purification And Activity Assays Of SIMO-Specific Protease 2(SENP2)

The requirement of soluble, correctly folded proteins in the field of lifescience presents tremendous challenges, the commonly used host for recombinant protein expression is E.coli, but it has many problem in over-express heterologous proteins,include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and so on. Gene fusions technologies can improve expression, solubility, and decrease proteolytic degradation.The primary disadvantage of fusion tag is the cleaving of the tag.But SUMO fusion tag has the advantage in the cleaving of the tag,because SUMO proteases can recognize the tertiary structure of SUMO tags and the cleavage generates natural N-extremities in the target protein. So the construction of the vector with SUMO fusion tag could be a choice when the other traditional vector can not express protein efficiently.As a fusion tag, ubiquitin can promote the soluble expression. pHUE (histidine-tagged ubiquitin expression vector), a ubiquitin fusion expression system which has been found by T. BAKER,can promote the solubility of the protein in E. coli. The protein can be simply purified by affinity chromatography. It has reported that yeast SUMO as fusion tags and molecular chaperones can increase the solubility of target proteins. Because human SUMO is highly homologous to yeast SUMO, so we can speculate whether hunman SUMO can act as fusion tags for soluble expression. It is more commendable that after cutting by the SUMO protease, amino acid residue is not resided. It is conducive to the subsequent experimental assays. According to the background, the development of a new type of soluble expression vector, will have a wide range of applications.In this paper, we conducted a human SUMO fusion protein expression system in promoting the solubility of the protein, and then we constructed two vectors pHSE1 and pHSE2 with human SUMO1 and SUMO2 tags respectively as fusion tags. By using two different target proteins, MMP-13 and GFP inserted into the vectors, to identification the increase of soluble target proteins in E. coli.We found that pHSE2 was better in promoting the solubility of the protein than ubiquitin taged vector pHUE, far greater than the pET vector. Therefore, we turned to the SUMO-specific protease SENP2's clone, expression and purification, to remove the SUM02 tag of the fusion protein expressed by PHSE2.And then the activity of target proteins was studied.The above results showed that the vector pHSE1and pHSE2, could promote the solubility of proteins.When the traditional technology can not obtain a valid expression of soluble protein, the vector pHSE1,pHSE2 which we constructed will be a good choice when united to use with the SUMO-Specific Protease SENP2.