In vitro activity of a recombinant ABC transporter protein in the processing of plantaricin E pre-peptide

Abstract

Most bacteriocins of lactic acid bacteria (LAB) are initially synthesized as pre-peptides with an N-terminal extension (leader peptides). Generally, the precursor peptides containing a double-glycine-type leader are processed by a dedicated ATP-binding cassette (ABC) transporter. The ABC transporter and an accessory protein lead to the cleavage of inactive pre-peptide with the concomitant export of the mature peptide across the cytoplasmic membrane. Plantaricins E, F, J, and K belong to class IIb 2-peptide bacteriocins and are synthesized as pre-peptides containing N-terminal G-G leader peptide. In this study, the heterologous expression, purification, and characterization of PlnE pre-peptide, ABC transporter (PlnG), and accessory protein (PlnH) from Lactobacillus plantarum LR/14 in Escherichia coli BL21 (DE3) strain were reported. An in vitro assay was conducted with the inactive PlnE pre-peptide, which after cleavage by the addition of ABC transporter protein exhibited antimicrobial activity against some LAB species. The activity of cleaved pre-peptide was comparable to the activity of mature peptide. Accessory protein was also heterologously expressed and purified; however, no effect on processing activity was detected by the addition of the accessory protein, which suggests that accessory protein is not involved in cleavage, but it might help in the transport of mature plantaricins across the membrane.

Identification of a two-component Class IIb bacteriocin in Streptococcus pyogenes by recombinase-based in vivo expression technology

“…Utilizing the MEROPS database46, S. pyogenes MGAS8232 contains a C39 peptidase motif within SilE, and an additional C39 peptidase motif in an ORF located ~5500 bp upstream of silA (locus tag SPYM18_RSO2285). The peptidase domain of bacteriocin ABC transporters are cytoplasmic47, and this domain can also function in isolation from the transporter48, so either gene could potentially provide machinery for leader cleavage of the Spb peptides. If operational, this ‘release by lysis’ mechanism would be functionally analogous to colicin release by E. coli 49.…”

Lactiplantibacillus plantarum–Nomad and Ideal Probiotic

“…The variety of bacteriocins produced by L. plantarum generally belong to Class II and are collectively referred to as plantaricins. Plantaricins may be either chromosomally or plasmid encoded and are usually organized within operon clusters that consist of a structural gene(s), an immunity gene, an ABC transporter and an accessory protein that facilitates export (Pal and Srivastava, 2015).…”

Heterologous Expression of Biopreservative Bacteriocins With a View to Low Cost Production

“…In addition, the use of fusion protein partners can increase the expression level, enhance protein solubility and assist in correct folding and disulfide bond formation (Ingham and Moore, 2007 ). Common fusion partners used for bacteriocin production include the cellulose binding domain (CBD cenA ) (Klocke et al, 2005 ), maltose-binding protein (MBP) (Quadri et al, 1997 ; Kim et al, 2006 ), thiorredoxin (Trx) (Gibbs et al, 2004 ; Richard et al, 2004 ; Beaulieu et al, 2007 ; Yildirim et al, 2007 ; Jasniewski et al, 2008 ; Caetano et al, 2011a ; Liu et al, 2011 ; Pal and Srivastava, 2014 , 2015a , b ; Jiang et al, 2016 ; Mustopa et al, 2016 ; Meng et al, 2017 ; Tang et al, 2018 ) and the small ubiquitin-related modifier SUMO (Wang et al, 2013 ).…”

“…The cyanogen bromide (CNBr) chemical cleavage strategy has been used to release the mature PlnE and PlnF (Fimland et al, 2008 ) and PlnJ and PlnK (Rogne et al, 2009 ), as has also been described for production of carnobacteriocin B2 and BM1 (Jasniewski et al, 2008 ) and piscicolin 126 (Gibbs et al, 2004 ). However, the most common approach to release recombinant bacteriocins is to include a sequence, between the signal peptide and the bacteriocin, recognized by Factor Xa (Quadri et al, 1997 ; Kawai et al, 2003 ; Klocke et al, 2005 ; Moon et al, 2006 ; Ingham and Moore, 2007 ; Rogne et al, 2008 ; Shi et al, 2011 ), trypsin (Shi et al, 2012 ; Himes et al, 2016 ), thrombin (Klocke et al, 2005 ), enterokinases (Beaulieu et al, 2007 ; Jasniewski et al, 2008 ; Liu et al, 2011 ; Pal and Srivastava, 2014 , 2015a , b ; Jiang et al, 2016 ; Meng et al, 2016 ; Tang et al, 2018 ), and SUMO proteases (Wang et al, 2013 ). Alternatively, the use of intein fusions has been described for the cloning and expression of self-cleaving fusion forms of unmodified bacteriocins under appropriate buffer conditions (Ingham et al, 2005 ).…”