Screening And Bioinformatic Analysis Of Heat Stress-resistant E.coli Strains Expressing Sea Cucumber Proteins

The imbalance of protein homeostasis(proteostasis)is one of the internal factors of aging and many diseases.It is known that heat stress response is directly associated with aging and proteostasis.As molecular chaperones,heat shock proteins can regulate the folding,assembly,transportation and degradation of other proteins,thus maintaining proteostasis.Caenorhabditis elegans has the advantages of short life cycle,typical aging phenotypes and high homology with human genes,and has become an important model for the study of aging and proteostasis.Using Escherichia coli clones from a sea cucumber(Apostichopus japonicus)cDNA expression library,here we first directly screened for heat stressresistant strains by feeding C.elegans.Then the gene sequences of the potential heat stress-resistant sea cucumber proteins were obtained by sequencing from the positive clones.Bioinformatic analysis was also performed to predict the structures,physicochemical properties and functions of the proteins of potential interest.The main results are as follows:1.Determination of high,medium and low survival rates of C.elegans population under heat stress.To accurately screen the protective effect of the expression library bacteria on the survival of C.elegans under heat shock,the heat stress(35?)and recovery(20?)times corresponding to high,medium and low survival rates(75%,50% and 25%)were determined using C.elegans fed on E.coli OP50,the routine bacterial food.When the nematodes were heat stressed at 35? for 16 h,the survival rate was about 60%,corresponding to a medium-to-low survival rate of the population,and therefore this heat stress time was selected for subsequent experiments.We also found that C.elegans would enter a “dormancy” state after surviving heat stress and need some time to restore its activity.A period of 12 h at 20? was sufficient for the nematodes to recover and thus was used as the recovery time for subsequent experiments.2.Effect of host bacteria of sea cucumber cDNA library on the growth and survival of C.elegans under heat stress.As food,different bacterial strains may have different effects on the growth,development and stress response of C.elegans.Therefore,we tested the effects of the host strain BL21(DE)plys S on C.elegans in comparison with E.coli OP50 and NA22,two commonly used bacterial food in laboratory,and found no significant difference in growth and heat stress survival as demonstrated by food clearance and heat stress survival assays,respectively.In addition,it is possible that difference in bacterial growth rate may have different effect on the nematodes,e.g.potential dietary restriction caused by slow growth of bacteria.We thus compared the growth rates of the library expression bacteria and the control host bacteria and found no significant difference.These results indicate that the host bacteria can be used as a control in subsequent heat stress experiments.3.Screening of heat stress-resistant strains expressing proteins from sea cucumber cDNA library.We first fed C.elegans with the expression library bacteria,and then tested the effect of the library clones on the survival rate of C.elegans under heat stress.This first round of screening generated 53 strains with potential activity against heat stress from 94 strains of the cDNA library clones.After further screening and verification,16 positive clones with heat stress resistance activity were obtained as candidate strains,the protein expression of which was confirmed by dot blot assay.4.Gene sequencing and blast analysis of candidate strains expressing potential heat stress-resistant proteins of sea cucumber.From the above positive clones,12 candidate strains were selected for gene sequencing,and the sequences obtained were subjected to BLAST analysis using the blastx algorithm with the species limited to Apostichopus japonicus.The sequences with the highest similarities were selected based mainly on Max Score and E-value but also on Identities and Gaps values.Through these analyses,six gene sequences were found in Gen Bank to have matching information with sea cucumber proteins.5.Prediction and analysis of the structure,physicochemical properties and functions of potential heat stress-resistant sea cucumber proteins.To further understand the properties and functions of potential heat stressresistant proteins,we performed bioinformatic analysis on the gene sequences.First,the primary structures of the potential heat stress-resistant proteins were obtained by blastx analysis,while the secondary and tertiary structures were predicted by PSIPRED Work Bench and SWISS-MODEL.Then the physicochemical properties of the potential proteins were predicted and analyzed using the Prot Param software to obtain such information as half-life,isoelectric point and instability coefficient.Functional prediction of the proteins was also performed using the BIOPEP-UWM software and Peptide Ranker online tool,and potential antioxidant activity was found in a number of proteins or their peptide sequences.In summary,we first determined the experimental conditions for the survival assay of C.elegans under heat stress,and then screened sea cucumber cDNA expression library bacteria for clones with potential activity against heat stress using the BL21 host bacteria as a control.The gene sequences of potentially active proteins were obtained by sequencing from the candidate clones.The amino acid sequences of these proteins were then predicted by sequence comparison analysis,and bioinformatic approaches were used to predict and analyze the structures,properties and activities of the proteins,which provides a basis for future studies on sea cucumber proteins.The screening method via direct feeding of proteinexpressing bacterial strains,rather than isolated proteins,at organismal level also provides a novel model for efficient screening of bioactive proteins.