To check the biofuctional activity of purified human cystatin-C, we tried an antimicrobial activity experiment. First, we checked antimicrobial activity of transgenic chickens’ egg white against E. coli. (Figure 10A).
Compared with human cystatin-C 50ng/ml, the clear zone was small but accurate. It is assumed that the clear zone is small because the egg white is so large that the concentration of cystatin is diluted. Next, we examined the antimicrobial activity of the purified cystatin-C in TG egg white (Figure 10B). Compared with human cystatin-C 100ng/ml and 50ng/ml, there were no clear zone in purified cystatin-C. Since the antimicrobial activity experiment was conducted immediately after protein purification, It is thought that the modification of protein is unlikely to occur after purification. It is assumed that the cause is in the purification method. In other studies, The egg whites are pretreated before purification for remove ovomucin (Wang and Wu 2014). In future studies, we will add a new pretreatment method to purify human cystatin and then confirm the antimicrobial activity.
Figure 10. Antimicrobial activity of human cystatin-C in transgenic chickens’
egg white against E. coli
(A) Identification of TG egg white’s antimicrobial activity. 1 is human cystatin-C 50ng/ml, 2 is water, 3 is TG EW. (B) Identification of purified human cystatin-C’s antimicrobial activity. 1 is human cystatin-C 100ng/ml,
2 is human cystatin-C 50ng/ml, 3 is purified and dialysis human cystatin- C in TG egg white.
Discussion
We constructed a vector using non-viral piggyBac transposon system to produce transgenic chickens expressing human cystatin-C and confirmed that the vector works normally by transfection in DF1 cells and PGCs. PGCs transfected with the identified vectors were microinjected into embryos to produce germline chimeras. Transgenic chickens were selected by testcross analysis among the chimeras’ offspring. When we identified the expression of human cystatin-C in various parts of the transgenic chickens, it was detected in all parts. This indicates that transgenic chickens expressing human cystatin-C have been produced.
In general, chicken is livestock that produce eggs. Thus, the males are eliminated from the chick stage. In addition, even when used as a bioreactor, the male chickens were excepted because the recombinant protein produce in egg white. In this study, the CMV promoter was used to express human cystatin-C throughout the chicken body, rather than the egg white specific promoter. This allows human cystatin-C to be purified not only on egg white but also on muscles and other tissues. It has the advantage that the recombinant protein can be obtained even in the muscles of males without eliminating the males when producing transgenic chickens. Using CMV promoters will have a positive impact on the productivity of recombinant proteins, reducing the effort of screening
male and female.
Human cystatin-C purification must be accompanied for use as medicines or diagnostic kits. In a previous study, they introduced a method for purifying cystatin from chicken egg whites using affinity chromatography (Wang and Wu 2014). However, we attached His tag back of the human cystatin-C sequence when we constructed the vector, only human cystatin-C protein was purified selectively using Ni-NTA magnetic nanobead. The protein was purified from egg white and homogenized muscle. But the amount of protein purified from muscle was higher due to differences in quantity of egg white and muscle. But the easier form to purify was egg white. In fact, when the purification purity was identified through SDS PAGE, purified sample in muscle had not only human cystatin-C but also other proteins. This is considered to be a problem of the purification method, and since the purification kit that we use is using magnetic nanobead, it is more likely that unwashed proteins will elute together when receiving the purification solution. In the case of muscle, the tendency to lump together with the resin was observed. Egg white was also agglomerated together with the resin, but after freeze-drying, diluting in PBS and once or twice with a sieve, it was confirmed that the resin agglomeration greatly reduced.
Cystatin is a cysteine protease inhibitor that has been known to have antimicrobial and antiviral properties against microorganisms and viruses through several papers (Szpak, Trziszka et al. 2014, Luthra 2015, Pikula,
Smuzynska et al. 2017). Using this property, we are conducting antimicrobial activity experiment to verify the activity of purified human cystatin-C. Antibiotics are one of the most commonly used treatments for bacterial infections. Antibiotic resistant microorganisms continue to emerge as the abuse of antibiotics and the ability of microorganisms to adapt. Many studies have been conducted to prevent the production of antibiotic resistant bacteria or to effectively eliminate them (Li, Li et al.
2018, Luna, Ershova et al. 2019, Sorbara, Dubin et al. 2019). If this experiment shows that the purified human cystatin-C has the antimicrobial activity, we can suggest the possibility of using the human cystatin-C protein as a new antibiotic.
We were concerned whether cystatin’s role as cysteine protease inhibitor would affect the basic mechanism of the transgenic chicken, but our transgenic chickens were not particularly different from those of normal chickens. In other papers, cystatin-C protein was never fed or injected as a drug, but there were no cases of health abnormalities when cystatin-C overexpressed individuals were produced and confirmed. Rather, it has been found to be possible as a therapeutic agent for inhibiting cancer or for neurological diseases such as Alzheimer’s disease (Kaeser, Herzig et al. 2007, Yan, Zhou et al. 2015, Coronado, Barrios et al. 2017, Zou, Chen et al. 2017, Kaur, Gauthier et al. 2018, Li, Wang et al. 2018).
In addition, cystatin expressed by parasites can induce the production of IL-10, which can suppress allergy and intestinal inflammation (Schnoeller,
Rausch et al. 2008, Jang, Cho et al. 2011). It is also being studied as a potential treatment for collagen-induced arthritis (CIA) (Liu, Cheng et al.
2016). Therefore, it was found that cystatin-C can be purified and used as a therapeutic agent in human cystatin-C expressing transgenic chickens.
Cystatin-C is generally known to have a characteristic of being filtered by glomeruli so that the glomerular filtration rate (GFR) can be calculated from the serum concentration of cystatin-C. Cystatin-C can thus be used as a marker for kidney disease. Traditionally, kidney function was assessed by serum creatinine concentrations. However, various studies have concluded that serum cystatin-C shows higher sensitivity and specificity than serum creatinine (Onopiuk, Tokarzewicz et al. 2015) and that more accurate data can be obtained with both creatinine and cystatin-C than with creatinine alone (Stevens, Coresh et al. 2008). However, when compared to creatinine, the cost of cystatin analysis is three times higher than the enzyme creatinine test and 20 times more than the creatinine test using the jaffe reaction (Shlipak, Mattes et al. 2013). Since the main cost is reagents, cystatin analysis will be more common if the reagent price can be reduced. This study is expected to contribute to the reduction in cystatin assay reagent prices.
With the advancement of gene editing techniques into zinc-finger nuclease, TALEN and CRISPR-Cas9, it is easier than ever to modify, add or delete the genes we want. Methods of making transgenic animals have also evolved to make faster and cheaper than before, which is an
economic advantage when using transgenic animals as bioreactors. Not many transgenic animals will grow rapidly, be easy to manage and produce protein continuously. Through this study, we believe that chicken is the most suitable animal for these conditions. In addition, the transgenic chicken production technique used in this study could be of great commercial value in the recombinant protein market if it is incorporated into other useful proteins.
Conclusion
In this study, the results can be summarized in two ways. First, transgenic chicken production using a non-viral piggyBac transposon system. For existing commercialized transgenic chickens, transgenic chickens were produced using a lentiviral vector system. The way in which viral vectors are used can pose safety concerns. In this study, we successfully produced transgenic chickens using the non-viral piggyBac transposon system, which we believe will help in the production of other useful recombinant drugs.
Second, presenting a new method for the production of cystatin-C.
Cystatin-C is well known as a marker for kidney disease and is being actively researched as a therapeutic agent for neurological diseases.
Human cystatin-C, which is on sale, is produced from animal cells and costs about 970,000 won per 10μg. The use of transgenic chickens continues to cost less than traditional animal cell-based production systems for the production of recombinant pharmaceuticals. Therefore, the production of human cystatin-C from transgenic chickens could lower the production cost and contribute to the study of the potential of human cystatin-C as a therapeutic agent.
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