Park, Chaeri
(College of Pharmacy, Catholic University of Daegu)
,
Song, Yun-Kyoung
(College of Pharmacy, Catholic University of Daegu)
,
Kim, Young-Hyun
(National Primate Research Center & Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology)
,
Jung, Yena
(College of Pharmacy, Catholic University of Daegu)
,
Park, Young-Ho
(National Primate Research Center & Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology)
,
Song, Bong-Seok
(National Primate Research Center & Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology)
,
Eom, Taekil
(College of Applied Life Sciences, the Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University)
,
Kim, Ju-Sung
(College of Applied Life Sciences, the Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University)
,
Kim, Sang-Hyun
(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University)
,
Kim, Ji-Su
(National Primate Research Center & Futuristic Animal Resource)
,
Kim, Sun-Uk
,
Lee, Sang-Rae
,
Kim, Ekyune
Hyaluronidases enhance therapeutic drug transport by breaking down the hyaluronan barrier to lymphatic and capillary vessels, facilitating their tissue absorption. Commercially available hyaluronidases are bovine in origin; however, they pose risks such as bovine spongiform encephalopathy. The prese...
Hyaluronidases enhance therapeutic drug transport by breaking down the hyaluronan barrier to lymphatic and capillary vessels, facilitating their tissue absorption. Commercially available hyaluronidases are bovine in origin; however, they pose risks such as bovine spongiform encephalopathy. The present study aimed to develop a novel, highly active hyaluronidase and assess its function. Therefore, in order to find the most efficient active hyaluronidase, we produced several shortened hyaluronidases with partial removal of the N- or C-terminal regions. Moreover, we created an enzyme that connected six histidines onto the end of the hyaluronidase C-terminus. This simplified subsequent purification using $Ni^{2+}$ affinity chromatography, making it feasible to industrialize this highly active recombinant hyaluronidase which exhibited catalytic activity equal to that of the commercial enzyme. Therefore, this simple and effective isolation method could increase the availability of recombinant hyaluronidase for research and clinical purposes.
Hyaluronidases enhance therapeutic drug transport by breaking down the hyaluronan barrier to lymphatic and capillary vessels, facilitating their tissue absorption. Commercially available hyaluronidases are bovine in origin; however, they pose risks such as bovine spongiform encephalopathy. The present study aimed to develop a novel, highly active hyaluronidase and assess its function. Therefore, in order to find the most efficient active hyaluronidase, we produced several shortened hyaluronidases with partial removal of the N- or C-terminal regions. Moreover, we created an enzyme that connected six histidines onto the end of the hyaluronidase C-terminus. This simplified subsequent purification using $Ni^{2+}$ affinity chromatography, making it feasible to industrialize this highly active recombinant hyaluronidase which exhibited catalytic activity equal to that of the commercial enzyme. Therefore, this simple and effective isolation method could increase the availability of recombinant hyaluronidase for research and clinical purposes.
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문제 정의
Alternatively, the recombinant human hyaluronidase produced by expression of the human SPAM1 gene in COS cells requires purification steps and generally shows low activity. Therefore, in this study, we aimed to develop a recombinant hyaluronidase with high activity, low risk of contamination, and at an easy-to-achieve, high level.
Thus, our data obtained from the chimeric hyaluronidase construction suggest that the disulfide bonds involved in formation of the protein structure are important for the activity of the recombinant hyaluronidase enzyme. This study was performed to determine whether the newly developed recombinant hyaluronidase showed stable activity at room temperature; if so, the enzyme would have high commercial value. In conclusion, our results suggest that rbSPAM1C80-del-6xH has the potential to rapidly dissolve a high polymer HA-based substance, indicating its effective application in cosmetic surgery, in vitro fertilization, and drug delivery.
제안 방법
To investigate the high activity level of hyaluronidase, we first constructed three fragments with encoding the N- and C-terminal regions (Short 1, 2, and 3), and inserted them into the pCXN2 mammalian expression vector, based on GenBank accession number NM-214011.1 (Fig. 1A). We focused on ways of producing and commercializing the newly reconstituted bovine hyaluronidase, by removing the C-terminal region possessing a very low homology with the human counterpart.
4D). To further explore the characterization of hyaluronidase, hyaluronidase was analyzed by a hyaluronidase inhibition assay by adding 2-mercaptoethanol(2ME). When compared with positive control, there was no hyaluronidase activity in the sample with added 2ME, despite the slight hyaluronidase activity in the boiled samples (Fig.
대상 데이터
Six oligonucleotide primers were designed to amplify DNA fragments encoding various bovine SPAM1 regions (Table 1). PCR was performed with a PCR thermal cycler (Takara Bio Inc.
성능/효과
This study was performed to determine whether the newly developed recombinant hyaluronidase showed stable activity at room temperature; if so, the enzyme would have high commercial value. In conclusion, our results suggest that rbSPAM1C80-del-6xH has the potential to rapidly dissolve a high polymer HA-based substance, indicating its effective application in cosmetic surgery, in vitro fertilization, and drug delivery.
후속연구
It is uncertain why the chimera showed a lower enzyme activity than the wild type; however, the structural aspect of the reconstituted hyaluronidase protein might affect the chimera activity. However, further studies need to be performed to elucidate why the chimera showed a lower enzyme activity. The rbSPAM1C80-del-6xH hyaluronidase exhibited an activity level equal to that of the commercial bovine native hyaluronidase in the high polymer HA dispersal assay (Fig.
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