title: Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.
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.
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China.
authors.forename: Hanyu
authors.initials: H
authors.lastname: Wu
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China.
authors.forename: Dainan
authors.initials: D
authors.lastname: Cao
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China.
authors.forename: Tongxin
authors.initials: T
authors.lastname: Liu
authors.affiliation: Wuxi KGBIO Biotechnology Limited Liability Company, Jiangsu 214145, P. R. China.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China.
authors.forename: Xiaoxiang
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D002645:Chickens / Q000235:genetics*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D009928:Organ Specificity; D010063:Ovum / Q000378:metabolism*; D010449:Peptide Mapping; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D009113:Muramidase