Design Of Signal Peptide For Recombinant Protein Secreted By Bacillus Amyloliquefaciens

The signal peptide is a short peptide located at the N-terminal of the protein,and its length is generally about 30 amino acids.The structure of the signal peptide consists of three parts: a positively charged N region;a highly hydrophobic H region;and a C region with a cleavage site.Signal peptides have the ability to guide precursor proteins into the secretory pathway and inhibit the folding of nascent peptide chains.So far,signal peptides have been widely used in the fields of recombinant protein production,disease diagnosis,and drug design.Studies have shown that the application of signal peptides can effectively improve the expression efficiency of recombinant proteins,but it is not yet possible to match a suitable signal peptide for each exogenous protein.Signal peptide screening requires a lot of time and resource costs,so the rational design of signal peptides is of great significance in the fields of production and R&D.In this study,the common H-region amino acid sequence of the signal peptide was designed by molecular docking,and then the molecular dynamics simulation method was used to describe the binding process of the signal peptide and SecA protein from the molecular point of view,and verified that the designed signal peptide was used in starch dissolving.The ability of Bacillus to secrete alkaline protease,and finally combined with the results of molecular dynamics simulations,the reasons for the different ability of signal peptides to guide secretion are explained.First,143 signal peptide sequences of the Sec secretion pathway derived from Bacillus subtilis were collected through the NCBI database and Signal P series software.Secondly,the three-dimensional structural model was obtained by using the structure prediction software.According to its scoring system,123 signal peptide sequences were finally obtained.3D structural model.Finally,the peptide-protein docking method was used to obtain the binding conformation of the signal peptide and the SecA protein,and the analysis software was used to obtain the interacting residues in the signal peptide.After statistical analysis,four common H-region amino acid sequences were obtained:(1)IICIVLGLFAALI;(2)IICICLGLFAALI:(3)IICIVLGSFAALI;(4)IICICLGSFAALI.The H region of the signal peptide Vpr was replaced by four common sequences of H region,and the interaction mechanism between the four designed Vpr signal peptides and SecA protein was studied in detail by using molecular docking and molecular dynamics simulation.Molecular dynamics simulation results show that in the complex structure of signal peptide and SecA protein,the NBD1,IRA2,HWD and PBD domains of SecA protein are more flexible,and the N and C regions of the signal peptide are more flexible.The binding of the signal peptide to the SecA protein mainly relies on the hydrophobic interaction.The binding of different signal peptides has little effect on the hydrogen bonds in each domain of SecA protein,and there are large differences in the effect on hydrogen bonds between the various domains of SecA protein.Based on the results of shake flask fermentation,the ability of Vpr-T2 in the designed signal peptide H region sequence to direct the secretion of alkaline protease was similar to that of the original signal peptide Vpr.In addition,these two signal peptides have unique effects on the substrate molecules of SecA protein.