Recombinant Expression And Functional Analysis Of Cystein-rich Protein CRP1 From Poterioochromonas Malhamensis

Metallothionein?MT?is a small molecule protein widely found in animals,plants and microorganisms.It is rich in cysteine and plays a role in maintaining metal ion homeostasis and detoxification of heavy metal ions in cells.Structure and function of MTs exhibit particularity and diversity.Numbers of identified MTs are rapidly increased.However,the number of MTs identified was relatively small in algae.In this study,a new PmCRP1 gene was identified from Poterioochromonas malhamensis.The primary structure of PmCRP1 is similar to MT.In order to explore the function of PmCRP1,expression of the gene and function of encoded protein were analyzed.The experimental results are as follows:1.Bioinformatics analysis of PmCRP1:The PmCRP1 is 396 bp with no intron and encodes 131 amino acids.PmCRP1 is rich in cysteine,and the Cysteine residues arranged in CCC,CXCC,XCCX,CXC,and XXCXX?C is Cys;X is any amino acid residue other than Cys?pattern.The conserved cysteine arrangement is similar to metallothionein,but its amino acid sequence shows its unique characteristics and cannot be classified into the existing 15 metallothionein families.Therefore,we speculate that the protein belongs a new metallothionein family.2.Identification and expression analysis of PmCRP1:Different metal ions were used to induce expression of PmCRP1 in P.malhamensis.RTPCR showed that different concentrations of Cd²⁺,Cu²⁺,Zn²⁺,Pb²⁺,and Hg²⁺failed to induce PmCRP1 up-regulation.Therefore,the PmCRP1 is not directly regulated by metal ions.3.Fusion expression and functional analysis of PmCRP1 in Escherichia coli:In order to study the function of the PmCRP1 protein,we constructed the recombinant plasmid pSUMO-PmCRP1 and transformed it into E.coli BL21.Firstly,the induced expression conditions were optimized,and the induction temperature and time were determined to be induced at 16?overnight.The IPTG concentration was 0.2 mmol/L.The SUMO-PmCRP1 fusion protein was purified by Ni affinity chromatography and its stability and metal ion binding capacity were detected.SUMO-PmCRP1 can stably exist in 10 mmol/L PBS after heating in a 85?for 10 min.Fluorescence quenching analysis showed that SUMO-PmCRP1 can bind 10 Cd²⁺,9 Cu²⁺and 4 Zn²⁺.4.Metal ion tolerance analysis of recombinant E.coli:To further analyze the metal ion binding ability of PmCRP1 protein,we constructed two truncated PmCRP1 proteins,tru29PmCRP1 with 29 amino acids truncated at the N-terminus and tru51PmCRP1 with 14 and 27 amino acids truncated at the N-terminus and C-terminus.We carried out Cd²⁺,Cu²⁺,and Zn²⁺tolerance assays in recombinant E.coli BL21/pSUMO,BL21/pSUMO-PmCRP1,BL21/pSUMO-tru29PmCRP1,and BL21/pSUMO-tru51PmCRP1.The results showed that recombinant E.coli BL21/pSUMO-PmCRP1 expressing full-length PmCRP1 fusion protein had the highest tolerance to Cd²⁺,Cu²⁺,and Zn²⁺,indicating that PmCRP1 can increase the tolerance of recombinant E.coli to metal ions.And after truncating the N-or C-terminal domain,its binding ability to metal ions is reduced.In this study,a new CRP1 gene was first identified from P.malhamensis.Bioinformatics analysis and metal ion chelation experiments showed that PmCRP1 has the ability to bind metal ions and can improve the tolerance of recombinant E.coli to metal ions.It may be a new metallothionein.This experiment provides new data for the further exploration of new metallothioneins and their functions in P.malhamensis,and this new metallothionein can be used for bioremediation of heavy metal ions in natural environment.