Cloning and heterologous expression of the genes encoding L. amylovorus S-layer proteins
The expressed slp genes (see above) were amplified by PCR from the chromosomal DNA of the L. amylovorus strains, cloned as NcoI-XhoI –fragments in E. coli DH5αF', sequenced to verify the correct open reading frames, and expressed in E. coli BL21 (DE3) as C-terminal hexahistidine tag-fusions, as described in the pET system manual (Merck KGaA, Darmstadt, Germany) and as previously reported [33]. Recombinant S-layer proteins were purified in the presence of 4 M guanidine hydrochloride (GuHCl) with His Trap HP columns (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. The pooled protein fractions were dialysed against deionized water overnight at +4°C, centrifuged (15 000 g, 20 min, +4°C) and stored in aliquots at -80°C.