Prokaryotic Expression And Nuclease Activity Determination Of AtHARBI1-2 Protein

HARBI1 protein belongs to the DDE?Tnp?4 family of proteins,it is a complete derivation of an active Harbinger transposable enzyme in a common ancestor of vertebrates(osteoichthys)about 450 million years ago,the HARBI1 gene is the only vertebrate gene that is homologous to the known Harbinger transposons.The protein with the highest homology to HARBI1 is encoded by an autonomous Harbinger3?DR transposons,the involunter transposons derived from Harbinger has a significant preference for a palindrome target site of 17bp,this has never been seen in any other DNA transposons,therefore,it is predicted that HARBI1 is a site-specific endonuclease.This study was based on the functional analysis of Arabidopsis thaliana AtHARBI1-2 gene in the laboratory,After the expression and purification of AtHARBI1-2protein by Escherichia coli prokaryotic expression system,in vitro activity study was conducted to verify the endonuclease activity of AtHARBI1-2 protein and to determine the optimal biochemical conditions for its endonuclease activity,the main results are as follows:(1)The DDE?Tnp?4 family gene AtHARBI1-2 was cloned from Arabidopsis thaliana,The prediction results showed that Atharbi1-2 protein had no transmembrane domain and was mainly localized in the cytoplasm.(2)The expression vector of Escherichia coli p ET30-his-AtHARBI1-2 was transformed into BL21(DE3),using prokaryotic expression system,SDS-PAGE gel detection revealed that the protein size was about 65 k Da,the protein concentration was0.33mg/m L.(3)The AtHARBI1-2 protein has nuclease activity,the opimum digestion temperature was 30?;g DNA and circular plasmid p ET30-his can be cleaved in Arabidopsis,there is no specificity in the selection of substrate.(4)EDTA can affect the activity of AtHARBI1-2 protein,and it is speculated that metal ions are necessary for the activity of AtHARBI1-2 protein.Nuclease activity of AtHARBI1-2 protein depends on the divalent metal ion Mg²⁺.150m M NaCl inhibited the activity of AtHARBI1-2 protein nuclease.(5)The optimum reaction conditions of AtHARBI1-2 protein nuclase were as follows:temperature 30?,Mg²⁺concentration 10mmol/L,EDTA concentration>8.0mmol/L,NaCl concentration>100mmol/L,the enzyme activity of AtHARBI1-2 protein was inhibited.