Molecular Cloning And Functional Analysis Of Cathepsin B Of Euplotes Octocarinatus

Cathepsin B(CTSB)is a class of hydrolase enzymes belonging to the papain-like cysteine protease family,which is widely found in viruses,mammals and humans.CTSB catalysis is achieved by a catalytic center consisting of three conserved amino acids,cysteine,histidine and asparagine.The structure of CTSB consists of a signal sequence,a prosequence,and a mature sequence.In the mature sequence,CTSB has an additional insertion sequence,an occluding loop structure,which gives CTSB a carboxy dipeptidase(exopeptidase)activity other than endopeptidase.Euplotes octocarinatus is a freshwater ciliate with a nuclear dimorphism,a vegetative macronucleus and a reproductive micronucleus.At the translation level,a high frequency of programmed ribosomal frameshift and genetic codon reassignment has been reported in this organism.UGA is reassigned as a cysteine.At present,CTSB research mainly focuses on parasites and human tumor cells.In addition to proteolysis,CTSB is also involved in immune escape,tumorigenesis and other processes.In order to study the biological function of cathepsin B in Euplotes octoearinatus,the gene of cathepsin B in Euplotes octoearinatus was cloned and sequenced,and the properties of its expression products were studied.The main results are as follows1.Molecular cloning and sequence analysis of the Eo CTSB gene family of the Euplotes octoearinatus.Using the amino acid sequence of human CTSB as the query sequence,six sequences encoding Eo CTSB were found in the database of the Euplotes octocarinatus.Phylogenetic tree analysis showed that the six Eo CTSBs sequences were homologous sequences of CTSB.Then,Six Eo CTSBs genes were cloned and sequenced.Nucleic acid sequence analysis revealed that the number of amino acids encoded by the six Eo CTSBs genes was about 290,the protein size was about 32 KD,and the isoelectric point was less than 7.Amino acid sequence analysis revealed that six Eo CTSBs have three catalytically active sites.Eo CTSB-1 and Eo CTSB-2 do not have a signal peptide structure,and Eo CTSB-3 has a function of catalyzing a specific substrate.None of the six Eo CTSBs have an occluding loop structure.Studies have shown that the loss of the occluding loop structure causes the disappearance of the exopeptidase activity,but increases the activity of the endopeptidase.2.Study of the stop codon in the in-frame of cathepsin B-1 of Euplotes octoearinatus.By analyzing the six Eo CTSBs sequences,we found that there are stop codons TAA and TAG in the open reading frame of Eo CTSB-1.Sequence alignment suggests that they are not frameshift positions,but most likely an amino acid could be encoded in such position.We tried to validate it at the nucleic acid and protein levels,respectively.At the nucleic acid level,single-cell PCR technique was used to amplify the Eo CTSB-1 gene using the different strains of the Euplotes octocarinatus as a template.The results showed that TAA and TAG codons were also present in the Eo CTSB-1 ORF in other strains.In order to further explore whether this gene is a pseudogene or a functional protein encoding gene,we designed and prepared a specific antibody against Eo CTSB-1.The antibody was used to carry out the western blot experiments on the total protein of Euplotes octoearinatus,and then the presence of Eo CTSB-1 protein was detected by immunofluorescence assay and liposomal transfection experiment.The results suggested that Eo CTSB-1 gene could encode a functional protein.3.Expression purification and enzyme activity assay of Eo CTSB-1.In order to successfully express Eo CTSB-1 in E.coli,we first optimized the gene sequence and constructed the recombinant expression vector p ET-30a-Eo CTSB-1.The plasmid was transformed into expression competent cell E.coli BL21,and the expression was induced by IPTG.The recombinant protein His-Eo CTSB-1 was purified by Ni column affinity chromatography.Finally,we used the fluorescing substrate Z-Phe-Arg-AMC to detect its optimum temperature and p H as well as thermal stability and p H stability.The optimum temperature for Eo CTSB-1 was 35 °C and the optimum p H was 5.0.In summary,this study identified six Eo CTSBs genes in Euplotes octocarinatus database.The genes were cloned and sequences analyzed.Several stop codons TAA and TAG in the open reading frame of Eo CTSB-1 was detected.We investigated this phenomenon at the nucleic acid levels and protein level.In order to study the biological function of cathepsin B in Euplotes octoearinatus,the Eo CTSB-1 was used as the research object,and we constructed the recombinant expression plasmid p ET-30 aEo CTSB-1 and transferred into prokaryotic cells for expression.Affinity chromatography obtained higher purity Eo CTSB-1 protein,and the optimum temperature of Eo CTSB-1 was 35 °C using the substrate Z-Phe-Arg-AMC,and the optimum p H was 5.0.The results obtained in this study lay a foundation for further study of the complexity of the genetic code of Euplotes octoearinatus,and provide a theoretical basis for further exploration of the catalytic activity and cell biological functions of different Eo CTSBs.