Questions and Answers
Q: What is Lysobac™ ?
A: Lysobac™ is the trade name for a highly active recombinant human
lysozyme which is expressed and purified from an animal-free system. Lysobac
is a non-animal alternative for human or chicken lysozyme typically used
for bacterial cell lysis in bioprocessing, diagnostics, and life science
research. In the biopharmaceutical industry, it is frequently employed
to recover therapeutically important proteins and biologics expressed
in recombinant E. coli system.
Lysozyme (muramidase) hydrolyzes preferentially the ß-1,4 glucosidic
linkages between N-acetylmuramic acid and N-acetylglucosamine which occur
in the mucopeptide cell wall structure of certain microorganisms.
Q: Is the physico-chemical properties of Lysobac equivalent to
the native form of human lysozyme?
A: The amino acid sequence and physico-chemical properties of Lysobac
is similar toto the same as the native form of lysozyme (EC 3.2.1.17)
commonly found in human biological fluids and secretions. Mass spectrometric
and electrophoretic analysis indicated that the molecular mass and isoelectric
point of Lysobac is identical to the native form. The pH, temperature,
and protease stability of Lysobac is analogous to the native form. In
addition, the specific activity of Lysobac is comparable with the commercially
available human lysozyme.
Q: What are the advantages of using Lysobac in bacterial cell
lysis?
A: The following are the advantages of Lysobac over the animal-derived
lysozymes: a) Lysobac is an animal-free product and is free of infectious
agents of animal origin. It therefore offers a regulatory benefit for
therapeutic proteins and vaccines expressed in bacterial systems such
as E. coli, b) Lysobac exhibited four times higher specific activity and
provides five times more protein yield than a commercially available chicken
lysozyme in lysing Micrococcus or E. coli cells. Hence, Lysobac can increase
bioprocessing efficiency and reduce production costs, and c) Lysobac is
a recombinantly-expressed protein, and can offer minimal lot-to-lot variability,
and more consistent cell lysis capability as compared with the animal-derived
protein.
Q: What are the common procedures for lysing E. coli cells with
Lysobac?
The common extraction buffer for E. coli cells contains 0.2 mg/mL of
Lysobac in 100 mM Tris-HCl, 2 mM EDTA, 0.05% Triton X-100, pH 8. The
cell paste is suspended in extraction buffer and incubated for at least
15 min at room temperature. Alternatively, Triton X-100 at 1% and PMSF
at 1 mM can be added separately and the lysis solution is further incubated
for at least 15 min at 37 °C. In other cases, freeze-thaw cycles,
sonication, and mechanical cell disruptions are also used to complement
the lysozyme lysis protocol.
Q: How much Lysobac is needed for optimal bacterial cell lysis?
The concentration of Lysobac needed for optimal cell lysis and protein
recovery depends on various factors in the bioprocessing conditions, and
have to be determined empirically. Typically, Lysobac is added to the
cell protein extraction buffer at a range of 0.05 to 0.5 mg/L or approx.
7,000 to 70,000 U/mL.
Q: Can Lysobac be formulated with other cell lysis reagents ?
Yes, Lysobac can be formulated with any other proprietary powder blend
of detergent and enzymes optimized for bacterial cell lysis. It is highly
soluble and compatible with common extraction solutions.
Q: Is it possible to immobilize Lysobac onto a solid support?
Yes, it is possible to immobilize Lysobac onto chromatography resins
or hollow fibers to serve as a lytic enzyme reactor for microbial cell
lysis.
Q: Can Lysobac be directly added to cell culture media?
Yes, Lysobac can be added directly to the microbial culture media to
provide for fast and efficient total lysate, and allow a non-denaturing
in-culture protein extraction.
Q: What is the storage stability of Lysobac?
A. The real time stability study showed that the product retains its
protein structural integrity and enzyme activity for at least two years
at 4°C and 25 °C, and at temperature as high as 37 °C.
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