B. Sample preparation
3.8 Factor Xa cleavage of MBP Fusion Proteins
The removal of the MBP tag from recombinant peptides, leucocin A and pre-leucocin A was necessary, since each fusion construct did not exhibit antimicrobial activity. It was suggested that the presence of the MBP affinity tag obstructed proper folding of leucocin A and pre- leucocin A thereby preventing antimicrobial activity. The maltose binding tag was removed from fusion proteins, MBP-LcaA and MBP-preLcaA, by cleavage with Factor Xa. Factor Xa is a serine protease isolated from bovine plasma and cleaves after the arginine residue in its preferred cleavage site, Ile-(Glu or Asp)-Gly-Arg (Maina et al., 1988; Gardella et al., 1990).
According to the pMAL protein and purification manual (New England Biolabs), MBP fusion proteins contain the Ile-Glu-Gly-Arg recognition site at their fusion joints. Therefore, Factor Xa cleavage after the arginine residue in this recognition site of MBP-LcaA and MBP- preLcaA, results in the removal of the MBP affinity tag.
A test digestion of each fusion protein with Factor Xa was performed in order to determine the optimal time required for total cleavage of the MBP tag from leucocin A and pre-leucocin A. It was found that an incubation time of 24 hrs is adequate for complete removal of the affinity tag from the recombinant proteins. Once this was established the reaction was scaled up. Factor Xa cleavage of the MBP fusion proteins was analyzed by 15 % (w/v) SDS-PAGE, which is seen in figure 3.26. As already mentioned, MBP-LcaA is approximately 46.433 kDa, while MBP-preLcaA corresponds to a molecular weight of 49.088 kDa. Slight differences in size can be seen between each fusion protein in lanes 2 and 4 (uncut controls) of figure 3.26.
Under optimal conditions, MBP fusion protein cleavage by Factor Xa produces two products, which consist of the MBP tag and the recombinant protein/peptide (Jenny et al., 2003). From figure 3.26, it can be seen that Factor Xa cleavage of MBP-LcaA and MBP-preLcaA yielded two of the above mentioned products, which are labelled on figure 3.26. The maltose binding protein in lanes 3 and 5 corresponds to a molecular weight of ~42.5 kDa, while leucocin A (3.933 kDa) and pre-leucocin A (6.588 kDa) are migrating along the 25 kDa level. These peptides are cationic in nature and aggregate during pore formation. Reports have suggested that certain bacteriocins are able to aggregate in an SDS environment. A study by Osmanagaoglu et al. (1998), reported differences in the molecular weight of pediocin F up to 16.6 kDa. Similar results were seen by Bhunia et al. (1987), and increases in the molecular weight of pediocin PA-1 were attributed to the ability of these peptides to aggregate in a SDS
environement. This explains the high molecular weight seen for leucocin A and pre-leucocin A.
Figure 3.26. 15 % (w/v) SDS-PAGE analysis of MBP-LcaA and MBP-preLeuA following Factor Xa cleavage. Lane 1: BIORAD Precision Plus Molecular Weight Marker (BioRad, South Africa), lane 2: MBP-LcaA uncut control, lane 3: cleavage of MBP-LcaA with Factor Xa, lane 4: MBP-preLcaA uncut control, lane 5: cleavage of MBP-preLcaA with Factor Xa.
Size differences are clearly seen for MBP-LcaA and MBP-preLcaA uncut controls in lanes 4 and 6. Factor Xa cleavage generated two products, each of which are labelled. The high molecuar weight seen for leucocin A and pre-leucocin A is attributed to aggregation as already explained.
3.8.1 Inhibition assay using cleaved MBP fusion protein
Once the MBP tag was removed by Factor Xa cleavage, each recombinant peptide within the cleavage mixture was tested for antimicrobial activity. The cleavage mixture consisted of maltose binding protein, Factor Xa, and the peptide of interest (leucocin A or pre-leucocin A).
Activity was tested by a deferred inhibition assay with L. monocytogenes as the indicator strain. From figure 3.27, it is apparent that each of the cleavage mixtures did not exhibit any activity against Listeria, as zones of inhibition were not seen for either of the samples tested.
The positive control (labelled C), did inhibit the growth of Listeria as a distinct zone of inhibition is seen.
The results can be interpreted by examining the properties of the solution, which contained the cleavage products. During affinity chromatography, MBP-LcaA and MBP-preLcaA, were eluted in column buffer containing 10 mM maltose. Factor Xa cleavage was performed in the same buffer which consists primarily of salts. Control leucocin A was purified using high performance liquid chromatography and was eluted in a volatile solvent (acetonitrile containing 0.1 % trifluoroethanol (TFE )). The mode of action of bacteriocins depends on their solution structure as this plays a vital role in the three dimensional folding of these peptides. Fregeau Gallagaher et al. (1997) tested the effects of two media, namely doecylphosphocholine (DPC) micelles and 90 % TFE, on the three dimensional structure of leucocin A. Here, it was established that this peptide adopts a more defined structure with regards to the C-terminal α-helix in 90 % TFE. As already mentioned in chapter one, the C- terminal α-helix is vital for activity, specifically pore formation, and site directed mutagenesis in this region results in a decrease in antimicrobial activity. In a similar study by Kaur et al.
(2004) it was concluded that peptides such as sakacin P, pediocin PA-1, carnobacteriocin B2, and leucocin A adopt more stable structures in TFE compared to water. Peptides in TFE were shown to maintain a conserved amphiphilic α-helix in the C-terminal region and a β-sheet or random coil in the N-terminal region. Thus, it can be concluded that volatile solvents such as TFE and TFA, are more suitable for the antimicrobial activity of class IIa bacteriocins.
Figure 3.27. Deferred inhibition of Listeria monocytogenes using Factor Xa cleavage mixtures for MBP-LcaA and MBP-preLcaA fusion proteins. Both cleavage mixtures did not inhibit the growth of L. monocytogenes, as no zones of inhibition were produced. The positive control, however, did produce a zone of inhibition. Samples spotted are labelled as follows:
Cleaved MBP-preLcaA (A); cleaved MBP-LcaA (B); Control (C).