Gene Cloning And Functional Characterization Of A Molecular Chaperone ClpK With Protein Disaggregation/degradation Dual Activity

Clp/Hsp100 proteins belong to the AAA+superfamily.Together with other molecular chaperones and proteases,they controls the quality and quantity of intracellular proteins,regulate intracellular protein levels,and maintain the homeostasis of protein network.One subfamily member ClpG_GIhas shown a stand-alone superhigh activity of protein disaggregation.Based on the phylogenetic relationship of ClpG/ClpK and the distribution of their inhibited microbials,herein we cloned the gene EmClpK of a chaperone ClpK from the nasal mucus,and subsequently performed a functional characterization by using the approaches of Escherichia coli expression and Arabidopsis transgenics.The main results are as follows:1.Gene cloning and preliminarily functional analysis of EmClpKBased on the alignments of the ClpG_GIgene against Gen Bank nucleic acid databases,primers were designed to amplify the EmClpK gene from the metagenome of nasal mucus.This fragment was then recombinantly cloned into p ET32a（+）to generate the prokaryotic expression vector p ET（EmClpK）.Although EmClpK encodes a protein that is most closed to ClpK from Methylobacillus flagellatus,it is very likely to originate from a species of Pseudomonas aeruginosa prevalently found in environment.The recombinant EmClpK protein was of good solubility and thermostability,and exhibited an ability of disaggregase which can unfold,refold,and partially restore the activity of denatured malate dehydrogenase.In the coexpression experiment of two vectors in E.coli,EmClpK showed a disaggregating/solubilizing effect on the target protein Jc APX1,but made the target protein Met A not accumulated,so it might also has the protein degradation-associated activity.In addition,the bacterial dot-plating test of serial dilutions demonstrated that the overexpression of EmClpK could improve the heat resistance of its recombinant E.coli strain.2.Further verification of EmClpK activity by changing the expression mode and intensityIn E.coli,by introducing cis-coexpression,generating mutations to regulate the expression intensity of EmClpK,conducting constitutive expression,and using more poorly soluble target proteins（e.g.Ps Ptx D,rbc L）for coexpression,the protein disaggregation/degradation-associated activities of EmClpK were further confirmed,and might have a dosage effect.3.Overexpression of EmClpK can improve the heat tolerance of transgenic ArabidopsisWe further constructed the plant expression vector p BI（EmClpK）and obtained EmClpK transgenic Arabidopsis through inflorescence infiltration of Agrobacterium.EmClpK could be expressed at the m RNA level in the roots,stems,leaves,and siliques of transgenic Arabidopsis plants.After heat treatment,EmClpK transgenic Arabidopsis had a higher survival rate,faster recovery growth,lower leaf electrolyte permeability,and better cell membrane thermostability,as compared with wild-type plants.This indicates that EmClpK transgenic Arabidopsis is of higher heat tolerance.In summary,this work revealed the universal protein disaggregation activity and substrate-specific degradation-associated activity of EmClpK.Due to the high homology,EmClpK should have a stand-alone superior disaggregation activity similar to that of ClpG_GI,and can be used as a molecular chaperone to improve the soluble expression of many aggregation-prone target proteins.In addition,overexpression of EmClpK can endow plants with enhanced heat tolerance,which might provide a new research route to improve plant thermal tolerance by utilizing members of the Clp/Hsp100 family.