Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin
Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows.
The CTAB (cetyltrimethylammonium bromide) genomic DNA extraction method was modified to use lysostaphin
and the methodology described by Johnson et al (23).
Then, three to four bacterial colonies were suspended and incubated in 150 [micro]L sterile distilled water with lysostaphin
(1mg/mL) (Sangon, China) at 37[degrees]C for 1h.
Three to four bacterial colonies were suspended in 150gl sterile distilled water with lysostaphin
(1mg/ml) and incubated at 370C for 30 mins according to the kit manufacturer's instructions.
In addition, lytic enzymes such as lysozyme, mutanolysin, and lysostaphin
can be used individually or in combination to enhance lysis of difficult-to-lyse organisms (Yuan et al.
In contrast, the absorbance of all cultures treated with lysostaphin
(a specific lytic enzyme for Staphylococci) dropped drastically early in the incubation period (within the first hour).
Cocktails combining cell-wall-active enzymes with differing specificities such as lysozyme, achromopeptidase, mutanolysin, and lysostaphin
may be a viable solution, albeit thorough decontamination may be a necessity.
For the extraction of bacterial DNA, 5-10 colonies were suspended in 100 [micro]lLTE buffer before adding 5 [micro]L lysostaphin
The answer was yes and the team quickly focused on lysostaphin
, an enzyme secreted by non-pathogenic Staph strains, harmless to humans and other organisms, capable of killing Staphylococcus aureus, including MRSA, and commercially available.
PEGylation also reduced the generation of antibodies against lysostaphin
protocols included lysozyme, lysostaphin
, and achromopeptidase from Sigma-Aldrich; proteinase K from Invitrogen; and a pyrimidine-specific RNase mixture, DNAse free (RNase, DNAse-free from Roche).