Profinity immobilized metal affinity chromatography ni2 charged resin

The expression and purification of M. hyopneumoniae glutamyl aminopeptidase and leucine aminopeptidase have been described previously^{37 (link), 40 (link)}. Similarly, the mhj_0169 gene predicted to encode for a methionine aminopeptidase in M. hyopneumoniae was synthesized and cloned into the expression vector PS100030 by Blue Heron Biotech (USA) converting all in frame TGA codons, which encode for tryptophan in mycoplasmas, to TGG. The recombinant construct was transformed into BL21 (Invitrogen, USA) using standard protocols outlined in the manufacturer’s instructions. Polyhistidine tagged rMHJ_0169 was purified under native conditions using 50% slurry of Profinity immobilized metal affinity chromatography Ni²⁺-charged resin (Bio-Rad) as per the manufacturer’s instructions. The eluted protein was dialysed against PBS in 10 K MWCO dialysis tubing and stored at −20 °C.

The mhj_0461 gene was synthesized and cloned into the expression vector PS100030 by Blue Heron Biotech (USA) removing in frame TGA codons. In mycoplasmas, the TGA codon encodes for tryptophan, which results in truncated proteins when expressing Mycoplasma genes in E. coli [26 (link)]. In frame TGA codons were mutagenized to TGG (sequence in the electronic supplementary material) and the recombinant construct was transformed into BL21 (Invitrogen, USA) using standard protocols outlined in the manufacturer's instructions. Polyhistidine tagged rMHJ_0461 was purified under native conditions using 50% slurry of Profinity immobilized metal affinity chromatography Ni²⁺-charged resin (Bio-Rad) as per the manufacturer's instructions. Briefly, a cleared BL21 cell lysate was mixed with Ni²⁺ resin overnight at 4°C, loaded into a 10 ml column and washed twice with 4 ml wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8). Bound proteins were eluted in elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8), dialysed against PBS in 10 K MWCO dialysis tubing and stored at either 4°C or −20°C.