Expression of linear permutated variants from circular enterocin AS-48.
To confirm whether the head-to-tail circularization could be involved in the stability and activity of the circular bacteriocin AS-48, two permutated linear structural as-48A genes have been constructed by circular permutation. The absence of the leaderless linear AS(23/24) and AS(48/49) proteins in Escherichia coli, under all the conditions investigated, supports the idea that the circular backbone is important to stabilize their structure and also indicates the significance of a leader peptide. In fact, the approach taken in this study to generate linear permutated proteins fused to an appropriate partner was sufficient to prevent cellular proteolysis. In this case, the high expression levels found favour their intracellular accumulations as inclusion bodies, which after solubilization showed a propensity to aggregate, thus hindering the specific EK cleavage. This could explain the presence of active hybrid tagged proteins identified in this work. The conserved distribution of hydrophobic and hydrophilic surfaces in the hybrid proteins is responsible for the antibacterial activity. In addition, the opening of the AS-48 molecule between the residues G(23) W(24) connecting the α1/α2 helices, confers greater stability, suggesting that the sequence and/or the free amino acid in the polypeptide chain are critical aspects in the design of new variants.
Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, C/Fuentenueva s/n, Granada, Spain.
Manuel
M
Montalbán-López
Manuel
M
Martínez-Bueno
Eva
E
Valdivia
Mercedes
M
Maqueda
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D013293:Enterococcus faecalis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001430:Bacteriocins; D011993:Recombinant Fusion Proteins; C466236:enterocin AS-48, Enterococcus faecalis