Deletion Of RimJ In BL21(DE3) And Investigation On Substrate Specificity Of N~α-terminal Acetylase

N-terminal acetylation is one of the most important protein modification methods, the contents of this paper is to study the deletion of the acetylase rimJ will affect the temperature sensitivity on E.coli BL21 (DE3) growth, and the substrate specificity of the two prokaryotic acetylase RimJ and NAT.1,Construct a targeting vector with two 1000bp long homologous arms, and an assistant plasmid can express homing endonuclease and Red recombinase under induction by L-arabinose, and two PCR procedures was used to confirm the deletion of gene rimJ. to compare the different temperature maximum specific growth rate by measuring the growth curve of original strain and mutant strain under three different temperatures(25℃,37℃,42℃), and use t value to analyze the significant difference of specific growth rate; the form of the two strains under different temperatures was observed under high power microscope; the ability expressing foreign protein was analyzed by expressing Tβ4 in the two strains. The test results showed that the two strains had no significant difference in maximum specific growth rate, strain forms and foreign protein expression. The deletion of rimJ will not affect the temperature sensitivity on E.coli BL21 (DE3) growth.2,To get greater than 90%purity acetylase RimJ and NAT by Ni-NTA affinity chromatography, dialysis treatment. Select the eight kinds of N-terminal hexapeptide as the two enzyme's substrate, and detect which substrates will be acetylated by the two enzymes through spectrophotometry after colored by DTNB. Co-expressing RimJ or NAT with respectively Tβ4 fusion protein or ESAT-6. The two foreign proteins were obtained in high purity by Ni-NTA affinity chromatography and reverse high performance liquid chromatography, and the molecular weight of Tβ4 fusion protein and ESAT-6 was analyzed by Q-ToF mass spectrometry. The results showed that RimJ could acetylate Tα1,ESAT-6,S5 of three hexapeptide, and NAT could acetylate Tα1,Tβ4,ESAT-6,S5,S18,L12,EF-Tu of seven hexapeptide. RimJ and NAT can acetylate Tβ4 fusion protein in vivo. Two experiments show that the deacetylase NAT has broader substrate characteristics.