A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity

Abstract

Background: Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity. Results: A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA 2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM. Conclusions: Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.

Sequence edition of single domains modulates the final immune and antimicrobial potential of a new generation of multidomain recombinant proteins

“…We have recently described a novel and versatile strategy to create easily editable multidomain proteins 13 . The parental construct, named JAMF1, linked up the HDP human alfa defensin 5 (HD5), a bacterial binding domain (gelsolin) and an enzymatic antimicrobial peptide (sPLA 2 ), flanked by two aggregation-seeding domains (c-Jun and c-Fos leucine zippers fragments at N- and C-terminal, respectively) 13 . HD5 is the major antimicrobial defensin peptide of ileal Paneth cells whose mechanism of action is based on pore-forming activity in the bacterial cell wall.…”

“…The multidomain protein JAMF1 combined the HDP human α-defensin-5 (HD5), a bacterial binding domain (gelsolin), and an enzymatic antimicrobial peptide (sPLA 2 ), flanked by two aggregation-seeding domains (a fragment of c-Jun and c-Fos leucine zippers at N- and C-terminal, respectively) in a single polypeptide. We demonstrated that the resulting antimicrobial multidomain protein showed activity against both Gram-positive and Gram-negative drug-resistant bacteria 13 . However we did not study if the domains were switchable or editable in a manner that the final function of the polypeptide could be modulated as needed.…”

“…This option, however, comprises a tedious and cost-ineffective work of carrier protein removal. To overcome these limitations, our group has recently published a new strategy based on the synthesis of several antimicrobial proteins and peptides combined in a single polypeptide that codifies a multidomain antimicrobial protein named JAMF1 13 . This approach allows to recombinantly produce antimicrobial peptides of interest at good yields, with no toxicity for the producer bacteria, and without the need of adding non-functional carrier proteins 13 .…”

“…To overcome these limitations, our group has recently published a new strategy based on the synthesis of several antimicrobial proteins and peptides combined in a single polypeptide that codifies a multidomain antimicrobial protein named JAMF1 13 . This approach allows to recombinantly produce antimicrobial peptides of interest at good yields, with no toxicity for the producer bacteria, and without the need of adding non-functional carrier proteins 13 . The multidomain protein JAMF1 combined the HDP human α-defensin-5 (HD5), a bacterial binding domain (gelsolin), and an enzymatic antimicrobial peptide (sPLA 2 ), flanked by two aggregation-seeding domains (a fragment of c-Jun and c-Fos leucine zippers at N- and C-terminal, respectively) in a single polypeptide.…”

Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications

“…However, over the last 15 years it has been broadly proven that proteins forming IBs are biologically active [ 4 – 7 ]. The presence of native proteins forming such aggregates has prompted to assess their potential as biomaterials for a wide range of applications, including biocatalysis, tissue engineering, and antimicrobial and cancer therapies [ 7 – 10 ]. They offer important advantages over their soluble counterparts such as high stability [ 11 ], slow-release behavior [ 12 , 13 ], and production through cost-effective processes [ 14 ].…”