Replacement of Trifluoroacetic Acid with HCl in the Hydrophobic Purification Steps of Pediocin PA-1: a Structural Effect

Abstract

Trifluoroacetic acid (TFA) is a purification contaminant associated with pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of pediocin. Consequently, we propose a new pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of pediocin PA-1.

The Continuing Story of Class IIa Bacteriocins

“…The 54 -dependent transcription displays three specific features. First, 54 recognizes specific promoters with a particular consensus sequence, which is different from those recognized by the housekeeping 70 factor. Second, the promoter sequence is located at position Ϫ12/Ϫ24.…”

“…In the case of the sakacin A producer Lactobacillus sakei 706, which harbors a three-component regulatory system, it has been shown that it is not possible to induce bacteriocin production by adding the inducer peptide VOL. 70,2006 CLASS IIa BACTERIOCINS 567 when this production has ceased due to growth near 37°C (49,139). Temperature-based regulation thus seems to overrule the quorum-sensing mechanism (139).…”

“…70,2006 CLASS IIa BACTERIOCINS 569 no clear secondary structure and is suggested to be relatively flexible (99). Homology modeling of immunity proteins belonging to the same subgroup as EntA-im ( Fig.…”

Effects of oral l-carnitine supplementation on insulin sensitivity indices in response to glucose feeding in lean and overweight/obese males

“…To date, several reports regarding the TFA toxicity against cells, e.g., suppression of proliferation of osteoblasts, were reported (Cornish et al 1999). In fact, counter-ions may also affect the secondary structure of peptides and proteins (Gaussier et al 2002). The presence of different counter-ions can affect hydrogen-bonding network and alter its structure (Blondelle et al 1995) (Cinelli et al 2001).…”

“…Characterization of synthetic peptides, including conformational analysis, is routinely performed using IR and CD spectroscopy. However, a strong IR band at around 1670 cm −1 assigned to TFA which overlaps the amide I band of peptides (1600-1700 cm −1 ) may complicate analysis (Gaussier et al 2002). Several methods of peptide TFA − counter-ion exchange are described in the literature.…”