Abstract
Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.
Highlights
Antibiotics added to the diets of poultry and pigs can improve animal productivity, health and meat quality [1]
The development of transgenic technologies enables the generation of transgenic animals for the production of recombinant proteins
The foreign proteins expressed by animal bioreactors could process posttranslational modifications, compared with the recombinant proteins synthesized in microbial cells [44]
Summary
Antibiotics added to the diets of poultry and pigs can improve animal productivity, health and meat quality [1]. Known as muramidase, is a 1,4-β-N-acetylmuramidase that degrades the glycosidic bonds in the peptidoglycan of bacterial cell walls [7]. It is widespread in tears, saliva, blood serum, human and cow milk, and avian egg whites [8,9]. A crucial capacity of lysozyme is to protect against infections of bacteria, viruses, and fungi [10,11,12]. It could be used as PLOS ONE | DOI:10.1371/journal.pone.0146032. It could be used as PLOS ONE | DOI:10.1371/journal.pone.0146032 December 29, 2015
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