Effect Of Tag Proteins On RHF-Cystatin Activity

Introduction:Haemaphysalis flava is the dominant tick species in China.It bites cattle and carries a large number of pathogenic microorganisms,and seriously harms the animal husbandry and public health safety.Cystatins are proteins which can inhibit the activity of cysteine endopeptidase.According to amino acid sequences,they can be divided into four types,i.e.,Cystatin 1-4.At present,the Cystatins in ticks mainly belong to Cystatin1 and Cystatin2.The main difference between them lies in the presence of C-PW-C motif and a signal peptide at the C-terminal of Cystatin2.Cystatins are detected in salivary glands,ovaries,midguts and other organs,and play important roles in processes such as blood digestion,immune regulation,pathogen invasion and embryo development in ticks.The development of vaccine is considered to be the most promising means for the prevention and control of ticks and tick-borne diseases.In terms of vaccine development,screening feasible protein antigens is a critical step.However,the full length of most members of Cystatin family in H.flava（Hf-cyst）is incomplete and their functions have not been confirmed.In this study,we aimed to investigate the effect of r Cystatin from different prokaryotic expression vectors on their activities to lay a foundation for the functional research and application of Cystatins.Methods:1.Partial sequence of Cystatin with annotation Contig24081.5127 was selected from a self-built library.Primers for 5’and 3’ends were designed based on the sequence.The full length of the sequence was amplified by the rapid-amplification of c DNA ends（RACE）.Bioinformatics analysis of the obtained full-length sequence was carried out by means of MEME,Signal P5.0 Server and other tools.2.ORF of Hf-cyst2,Hf-cyst3,HF-cyst5 and Hf-cyst6 were inserted into p ET28a,and ORF of Hf-cyst6 was inserted into p ET32a and p GEX-4T-1.Positive plasmids were selected and transformed into BL21（DE3）.After culture for 12 h,positive colonies were selected and cultured.Inducer IPTG was then added to prepare recombinant proteins,i.e.,rHf-cyst2,rHf-cyst3,rHf-cyst5 and rHf-cyst6.Recombinant proteins were purified on commercial purification columns.Activities of the purified recombinant proteins were determined according to the manual of a Cathepsin S Inhibitor Screening Kit.Results:1.The amplified and spliced sequences were 684 bp in length,and the ORF length was 399 bp,encoding a polypeptide of 133 aa with 1-17 aa as a signal peptide.The analysis by MEME showed that the encoding protein had the Cystatin family 2 motif and should be Hf-cyst6.The sequence was uploaded to Gen Bank with the access number MW091499.2.SDS-PAGE showed that all the recombinant proteins were expressed as inclusion bodies,and only a small amount of soluble expression was found in rHf-cyst6 expressed by p ET32a and p GEX-4T-1.The p ET28a expressed rHf-cyst2(rHf-cyst2-His²⁸),rHf-cyst3(rHf-cyst3-His²⁸),rHf-cyst5(rHf-cyst5-His²⁸),rHf-cyst6(rHf-cyst6-His²⁸),p GEX-4T-1 and p ET32a expressed rHf-cyst6(rHf-cyst6-His³²,rHf-cyst6-GST)all inhibited the activity of Cathepsin S after denaturation,purification and renaturation.The trend of the 7 recombinant proteins was consistent,that is,the inhibitory effect was enhanced with the increase of the concentration.Specifically,rHf-cyst5-GST,rHf-cyst5-His²⁸,rHf-cyst3-His²⁸,rHf-cyst2-His²⁸,rHf-cyst6-His²⁸,rHf-cyst6-His³²,rHf-cyst6-GST inhibited the Cathepsin S at a concentration of 0.25mg/m L,0.25mg/m L,0.0625mg/m L,0.05mg/m L,0.5mg/m L,0.2mg/m L,0.16mg/m L,respectively.Conclusion:1.Bioinformatics analysis indicated that Hf-cyst6 belonged to the Cystatin family 2.2.The activity of rHf-cyst is affected by the tag protein.Among the four proteins expressed by p ET28a,rHf-cyst2-His²⁸ had the highest inhibitory effect.For rHf-cyst6,rHf-cyst6-GST had the highest inhibitory effect.rHf-cyst5 was expressed by p GEX-4T-1 and p ET28a,and the inhibition effect of rHf-cyst5-GST was stronger than that of rHf-cyst5-His²⁸.The results of this study provide a basis for the selection of expression vectors.