Conversion Of Outer Membrane Protein A Signal Peptide Into Antimicrobial Peptide

OBJECTIVE:To explore the possibility of conversing the Escherichia Coli(E.coli)outer membrane protein A(OmpA)signal peptide into antimicrobial peptides,and to provide a new direction for the research of new antimicrobial agents.METHODS:(A)the primary structure of E.coli OmpA signal peptide MKKTAIAIAVALAGFATVAQA(referred to as:P-AQA)as a template,the C-terminal region amino acid sequence TVAQA were replaced by three or five amino acids.The following three derived peptides were synthesized as follows:(1)Derived peptide 1:MKKTAIAIAVALAGFARVRQK(abbreviation:C-RQK),the sequence TVAQA were replaced by three basic animo acid resides,thesequenceasRVRQK;(2)Derivedpeptide2:MKKTAIAIAVALAGFARRKKK(abbreviation:C-KKK),the sequence TVAQA were replaced by five basic animo acid resides,the sequence as RRKKK;(3)Derived peptide 3:MKKTAIAIAVALAGFADVDQD(abbreviation:C-DQD)the sequence TVAQA were replaced by three acidic amino acid D,the sequence as DVDQD;(B)In addition to the C-terminal region of the conversion,took derived peptide 2(C-KKK)as a template for the intermediate hydrophobic region(H region)of the amino acid increased or decreased,residued the following three peptides:(1)Derived peptide 4:MKKTA....VALAGFARRKKK(abbreviation:H-IAI)four of the hydrophobic amino acids IAIA in the amino acid AIAIAVALAGFA of the C-KKK region H were removed,the sequence as AVALAGFA;(2)Derived peptide 5:MKKTANAIAVALAGFARRKKK(abbreviation:H-NAI)one of the hydrophobic amino acids I in the amino acid AIAIAVALAGFA of the C-KKK region H were replaced by neutral amino acids N,the sequence as ANAIAVALAGFA;(3)Derived peptide 6:MKKRTLLLLLLLLLGFARRKKK(abbreviation:H-LLL)nine hydrophobic amino acids of(H region)AIAIAVALAGFA were replaced by hydrophobic amino acids L,at the same time a basic amino acid R was added at the N-terminus,the sequence as LLLLLLLLLGFA;(C)in addition to the C-terminal region and H regiones were transformed,ensure that the N-terminal basically structure was no changed,we increase basic amino acids in the N-terminal region signal peptides,at the same time,residued the following three peptides:(1)Derived peptide 7:MKKRRKTAIAIAVALAGFATVAQA(abbreviation:N-RRK)took P-AQA as a template,added 3 basic amino acid RRK at the N-terminus regione;(2)Derived peptide 8:MKKRTAIAIAVALAGFARRKKK(abbreviation:N-RTA)took derived peptide C-KKK as a template to add a basic amino acid R at its N-terminus(.D)In order to understood the importance of ensuring the structural integrity of the derived peptide,we designed and synthesized a derived peptide consisting of 10 basic amino acids,named,the derived peptide 9:KKRRKKRRKK(abbreviation:P-RKK)as the control peptide.The bactericidal activity of different signal peptide derived peptides was determined by agar plate count method.9 different derived peptides with different concentration gradients were interacted with Gram-positive(G~+)bacteria Staphylococcus aureus and Gram-negative(G~-)bacteria E.coli in reaction solution,After incubation at 37°C for 2 hours,the reaction solution,were diluted and incubated in a 37°C incubator for 16-18hours.Colony-forming units(CFU)were counted and calculate the sterilization of different derived peptides.RESULTS:Natural E.coli OmpA signal peptide is insoluble in water and can not detect its bactericidal activity.(1)C-RQK,C-KKK have strong bactericidal activity against both Staphylococcus aureus and E coli.The bactericidal activity was 99.9%at the concentration of 200ug/ml,and the bactericidal activity is C-KKK>C-RQK.(2)The bactericidal activity of P-RKK to Staphylococcus aureus is lower than that of C-RQK and C-KKK,Almost no bactericidal activity on the E coli.C-DQD has no bactericidal activity against Staphylococcus aureus and E coli.Activity is C-KKK>C-RQK>P-RKK>C-DQD.(4)The amino acid composition(H-LLL)of the intermediate hydrophobic group is changed by C-KKK as a template,which has no effect on its bactericidal activity,emoval of a part of the hydrophobic amino acid(H-IAI)or replace one of hydrophobic amino acid into a neutral amino acid(H-NAI)has a big chang to there bactericidal activity of Staphylococcus aureus and E coli,almost no bactericidal effect.(5)with E.coli OmpA signal peptide as a template in the N-terminal region to add three basic amino acid design of synthetic peptide N-RRK on Staphylococcus aureus almost no bactericidal activity of E.coli has a weak bactericidal effect to C-KKK The bactericidal activity of N-RTA on Staphylococcus aureus and E coli is almost no difference from that of C-KKK at the N-terminal.CONCLUSION:(1)The bactericidal activity of the E.coli OmpA signal peptide-derived peptide is positively correlated with the basic amino acid in the C-terminal region.(2)The intermediate hydrophobic structure plays a vital role in the bactericidal activity of the derived peptide,shortening the hydrophobic amino acid composition or reducing the bactericidal activity of a hydrophobic amino acid to a neutral amino acid and then reducing the bactericidal activity,but only to change the composition of its hydrophobic amino acid has no effect on its bactericidal activity.(3)Changing the N-terminal region has little effect on the bactericidal effect of the signal peptide-derived peptide.