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Secretory expression and scale-up production of recombinant human thyroid peroxidase via baculovirus/insect cell system in a wave-type bioreactor.
Lou, Ying; Ji, Guorong; Liu, Qin; Wang, Pengbo; Zhang, Ruilin; Zhang, Yuanxing; Liu, Xiaohong.
  • Lou Y; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, China.
  • Ji G; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, China.
  • Liu Q; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, China; Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai, China.
  • Wang P; Shanghai Wei Sheng Marine Biotechnology Co., Ltd., Shanghai, China.
  • Zhang R; School of Life Sciences, Fudan University, Shanghai, China.
  • Zhang Y; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, China; Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai, China.
  • Liu X; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, China. Electronic address: liuxiaohong@ecust.edu.cn.
Protein Expr Purif ; 149: 7-12, 2018 09.
Article en En | MEDLINE | ID: mdl-29655787
ABSTRACT
The human thyroid peroxidase (hTPO) is an essential enzyme for thyroid hormone biosynthesis and is expressed in thyroid cells. It is an autoantigen against which antibodies are found in the sera of patients with a number of autoimmune thyroid disorders. Overexpression of hTPO has been achieved using the baculovirus expression vector system (BEVS). However, it is produced largely in an aggregated form in the cell lysate fraction, which increases the complexity of protein extraction. In this study, to achieve improved secretory expression of hTPO via BEVS, a truncated recombinant hTPO protein (hTPOt) was engineered by replacing its original signal peptide (SP) in the N-terminal with five heterologous SPs. Our data showed that the SP from the peptidyl-glycine alpha-amidating monooxygenase (PAM), referred to as SPPAM, significantly promoted the secretion of SPPAM-fused hTPOt (p-hTPOt) in High Five cells. Subsequently, we established an optimized scale-up production procedure for p-hTPOt in a 5-L wave-type bioreactor. The secretory p-hTPOt was purified by immobilized metal-chelating affinity chromatography and ion-exchange chromatography, achieving a protein purity of >95%. Finally, the purified p-hTPOt showed high sensitivity and specificity in reactions with positive or negative human serum samples via the double-antigen sandwich method, suggesting potential applications in hTPO-based research and product development.
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Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Autoantígenos / Reactores Biológicos / Proteínas de Unión a Hierro / Yoduro Peroxidasa Límite: Animals / Humans Idioma: En Año: 2018 Tipo del documento: Article

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Autoantígenos / Reactores Biológicos / Proteínas de Unión a Hierro / Yoduro Peroxidasa Límite: Animals / Humans Idioma: En Año: 2018 Tipo del documento: Article