title: High level expression, efficient purification, and bioactivity of recombinant human metallothionein 3 (rhMT3) from methylotrophic yeast Pichia pastoris.
abstract: Metallothionein 3 (MT3) is an important biochemical mediator regulating many physiological and pathophysiological processes including neuron cell protection, privation of reactive oxygen species-induced DNA damage, and protection against light induced retinal damage. In this study, a human gene encoding for MT3 with c-terminal extension of His6-tag was inserted into vector pPICZaA, and overexpressed in Pichia pastoris strain X-33. The rhMT3 was purified by one step Ni(+)-NTA affinity chromatography yielding 270mg/L of over 90% purity. Functional analysis of the purified rhMT3 using inductively coupled plasma mass spectrometry demonstrated that it has biological function, binding with metal ions Cd(2+), Cu(2+) and Zn(2+). In summary, the experimental procedure we have developed facilitates production of large amounts of an active rhMT3 for further research and drug development.
authors: Department of Anatomy, School of Medicine Shandong University, Jinan 250012, China.
authors: Qi
authors: Q
authors: Wu
authors: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China.
authors: Bin
authors: B
authors: Li
authors: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China.
authors: Fei
authors: F
authors: Wu
authors: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors: Lijun
authors: L
authors: Yang
authors: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors: Shiwu
authors: S
authors: Li
authors: The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors: Hongbo
authors: H
authors: Li
authors: The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors: Donghai
authors: D
authors: Wu
authors: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China.
authors: Taixing
authors: T
authors: Cui
authors: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China. Electronic address: tangdq@sdu.edu.cn.
authors: Dongqi
authors: D
authors: Tang
mesh_terms: D002104:Cadmium / Q000737:chemistry; D002846:Chromatography, Affinity; D003300:Copper / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry; D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D051964:Receptors, Mating Factor / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D015032:Zinc / Q000737:chemistry
chemical_list: D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D051964:Receptors, Mating Factor; D011994:Recombinant Proteins; D002104:Cadmium; D003300:Copper; D015032:Zinc
keywords: High-density fermentation; Human metallothionein 3; Metal ions adsorption; Pichia pastoris