Production of human hyaluronidase in a plant-derived protein expression system: plant-based transient production of active human hyaluronidase.
Four types of human hyaluronidases (rHuHyal-1, -2, -3 and -4) were transiently expressed and purified from Nicotiana benthamiana, and their biochemical characteristics were analyzed. The recombinant HuHyals were expressed via agrobacteria-mediated infiltration and generated and expressed in terms of micrograms per 5 leaves of N. benthamiana. Expressed recombinant HuHyals were purified using a His(6) tagging system and Ni column chromatography, respectively, at pH 8.0, after which the purified rHuHyals were concentrated for additional biochemical analyses. The four types of rHuHyals were allowed to react with hyaluronic acids and chondroitin sulfates. The biochemical properties of rHuHyal-1 fit those of the commercially available Hyal, PH-20, which was extracted from animal testes under acidic conditions (pH 3.5). However, rHuHyal-1 evidenced activity levels 2 to 6-fold greater than the three other rHuHyals (rHuHyal-2, -3 and -4) at pH 3.5. However, only rHuHyal-4 exhibited chondroitinase activity with both 6-S-chondroitin sulfate (chondroitin sulfate C) and 4-S-chondroitin sulfate (chondroitin sulfate A) as standard substrates. The results of zymography demonstrated that recombinant HuHyal 1 was modified by glycosylation, but Escherichia coli Hyal was not. This result demonstrated that plant-based rHuHyal was functionally active and evidenced biochemical characteristics and post-translational protein modifications similar to those of animal testis-derived Hyal.
Jung
Yuchul
Y
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Republic of Korea.
Jung
Man-Yong
MY
Park
Jin-Hee
JH
Jung
Gyou Chul
GC
Hong
Young Seon
YS
Yeom
Chang Hwan
CH
Lee
Sukchan
S
D003001:Cloning, Molecular; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D006821:Hyaluronoglucosaminidase