| Most of the proteins in organisms must be folded correctly for a native conformation to achieve their biological activities.The nascent polypeptides are prone to misfolding,leading to formation of aggregates with potential toxicity to cells.Therefore,it is vital to maintain protein homeostasis for the health of cells or full body.Chaperones are involved in the translation,folding,unfolding,translocation and degradation of proteins,in which the DnaK-DnaJ-GrpE(KJE)system prevalently plays an important role in living domains of animals,plants and microbes.As the crucial constituent of this chaperone system,DnaJ is susceptible to dysfunction especially under stress conditions.Consequently,misfolded proteins can not be efficiently protected from aggregation,and even sequester chaperones into coaggregates,leading to a number of biological abnormalities such as inclusion body formation,amyloid-related neurodegenerative diseases.Therefore,it is necessary and of interest to potentiate DnaJ activity by molecular manipulation with an emphasis on the aspect of protein solubility.At present,fusion expression strategy,using solubility-enhancing adapters(especially hyper-acidic minipeptide),has been emerged as an important means to enhance protein solubility and thermostability.In this study,we selectively use two effectual hyper-acidic minipeptides(ra3t,tua2)as fusion partners,aiming to improve the solubility of Escherichia coli chaperone DnaJ and further the chaperoning activity.The major results are shown as follows:1.Vector construction(1)Construction of prokaryotic expression vectors containing chaperone genesE.coli DnaK(K),DnaJ(J)and GrpE(E)were amplified from the genomic DNA of E.coli BL21(DE3)strain,then subclond into plasmid pET32a(+)between NdeI and XhoI by enzyme digestion to obtain prokaryotic expression vectors pET(K),pET(J)and pET(E),respectively.(2)Construction of DnaJ fusion expression vectorsOn the basis of pET(J),a series of DnaJ fusion expression vectors were constructed by in-frame fusing the hyper-acidic minipeptides,ra3 t and tua2,at the N-terminal,C-terminal or both ends of DnaJ.(3)Construction of prokaryotic expression vectors containing target genesThe coding fragments of JcAPX1(APX1),EcMetA(MetA),JcTBP1(TBP1),NtRCA(RCA)and DnaJ(J)genes were re-amplified from their preexisting pET constructs,to create a new set of pACYC184-background expression vectors by recombinant cloning,including pAY(APX1),pAY(MetA),pAY(TBP1),pAY(RCA)and pAY(J).The NtrbcL(rbcL)gene and the DNA fragment encoding BoWSCP(WCP)mature chain were amplified from the genomes of tobacco and cauliflower,respectively.Meanwhile,the coding region of EGFP(GFP)was reamplified using plasmid pEGFP-N1 as template.Finally,these PCR fragments were used to construct prokaryotic expression vectors pAY(rbcL),pAY(WCP)and pAY(GFP)by recombinant cloning.2.DnaJ is of high priority for functional potentiation within the KJE chaperone systemThe recombinant DanK and GrpE proteins commonly had a high solubility of nearly 90%,while the recombinant DnaJ was of relatively low solubility(about 50%),suggesting that DnaJ has a necessity and possibility for functional potentiation,prior to other components of KJE chaperone system.3.Tailing hyper-acidic minipeptides improves DnaJ solubilityThe solubility of DnaJ could be significantly enhanced by at least 1.4-fold,when tailing the hyper-acidic minipeptides,ra3 t and tua2.4.Coexpression analysisThe recombinant coexpression in E.coli is feasible for chaperone-harbored pET serial vectors and the pAY serial vectors containing various target genes.Herein,this strategy was used to assess the influence of the chaperones on their co-expressed target proteins,in terms of protein solubility and activity.(1)DnaJ fusion with hyper-acidic minipeptide can further improve the solubility and enzymatic activity of APX1DnaJ and all of its fusions with hyper-acidic minipeptides could improve thesolubility of coexpressed APX1,in which the C-terminal fusions were dominant and thus used for subsequent studies.In addition,both in-gel activity staining and enzymatic assay showed that the hyper-acidic DnaJ fusions could convey APX1 a better enzymatic activity than DnaJ itself.This result is consistent with the scenario of solubility enhancement,implying that the hyper-acidified DnaJ is effectual to assist formation of the authentically soluble APX1 with correct folding and normal function.(2)DnaJ fusion with hyper-acidic minipeptide can further enhance the solubility and fluorescence activity of GFPIn comparison to the native DnaJ,the hyper-acidic DnaJ fusions could further improve the solubility of coexpressed GFP,and the fluorescence excitation profiles additionally demonstrated that it could also increase GFP fluorescence to a larger extent.This data implies that the hyper-acidified DnaJ is effectual to facilitate formation of the authentically soluble GFP with correct configuration and normal function.(3)DnaJ fusion with hyper-acidic minipeptide can further improve the solubility of MetA and the heat tolerance of the corresponding E.coli strain for recombinant coexpressionAs compared to the native DnaJ,the hyper-acidic DnaJ fusions could more significantly improve the solubility of coexpressed MetA.Moreover,bacterial dot-plating tests showed that the E.coli strain with recombinant coexpression of hyper-acidic DnaJ fusion and MetA was of the best growth status on M9 basal medium under a heat stress of 44 ?,indicative of a relatively highest heat tolerance conveyed by hyper-acidified DnaJ to E.coli cells.Conclusively,these results considerably suggest that the hyper-acidified DnaJs are indeed effectual to favor formation of the authentically soluble MetA with correct folding and normal activity,and endue E.coli cells with a predominant heat tolerance.(4)DnaJ fusion with hyper-acidic minipeptide can further improve the solubility of other target proteinsAdditional investigations showed that the hyper-acidic DnaJ fusions could more significantly enhance the solubility of other coexpressed target proteins includingrbcL,RCA,WCP,and TBP1,as compared to DnaJ itself.5.DnaJ fusion with hyper-acidic minipeptide can further improve the heat tolerance of E.coli and yeast strains without coexpressionBacterial dot-plating tests showed that the recombinant E.coli strain of hyper-acidic DnaJ fusion grew better than that of DnaJ on LB medium under 44 ? heat stress.Meanwhile,yeast dot-plating tests also demonstrated that the recombinant Saccharomyces cerevisiae strain of hyper-acidic DnaJ fusion was of a relatively better growth at 51 ? on YPD medium,as compared to the yeast strain of DnaJ.These data indicate that the hyper-acidified DnaJs confer an increased universal chaperoning activity,and contribute to their recombinant E.coli and yeast strains with a relatively higher tolerance to heat.6.DnaJ fusion with hyper-acidic minipeptide can improve the solubility of itselfDnaJ,using as a target protein,was affected with at least 1.2-fold increase in its solubility,when coexpressing with the hyper-acidic DnaJ fusions.This finding implies that DnaJ can be improved by its hyper-acidified cognates in respects of the solubility and probably the activity.In all,our above results indicate that DnaJ is successfully potentiated with an increased chaperoning activity,just by tailing hyper-acidic minipeptides.Comparing to the native DnaJ,the hyper-acidified DnaJ can benefit its target proteins with higher solubility and activity,and even endue the recombinant E.coli and yeast strains with better heat tolerance.The hyper-acidic DnaJ fusion has a higher solubility than its native form,probably due to the introduced force of negative charge repulsion.Resultantly,it is more potent to bind client proteins into the soluble phase for a more efficient prevention of aggregation,thus favoring the correct folding of target proteins.On the other hand,DnaJ solubility can be improved by its hyper-acidic fusion cognates,which is certainly helpful for its chaperoning activity.In our assumption,above two mechanisms could coexist,but the former one might be the dominant. |
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