title: A convenient method for hTfR1 inclusion body purification.
abstract: Human transferrin receptor, referred as hTfR1, is ubiquitously expressed at low levels in most normal human tissues; however, the expression level of hTfR1 at the blood-brain barrier (BBB) and in tumor tissues is relatively higher. hTfR1 is a type II homodimeric transmembrane protein. The extracellular domain of hTfR1 consists of three domains: helical domain, apical, and protease-like domain. In order to prepare hTfR1 antibody, which can be utilized to deliver drugs across BBB through receptor-mediated endocytosis, we began to express the nonligand binding domain of hTfR1 in Escherichia coli BL21 Transetta (DE3). The TfR1 gene was first obtained from HepG2 cells by reverse-transcription polymerase chain reaction (RT-PCR) and then inserted into pET 32a(c+) vector. The protein was expressed in the form of inclusion body with extremely high purity by the E. coli BL21 Transetta (DE3), and the purity was further improved by size-exclusion chromatography. The Western blot test indicated that the recombinant protein was TfR1 as expected. Above all, this report provided a convenient protocol that could be fulfilled in order to prepare hTfR1 inclusion body, which failed to be purified by an Ni(2+) affinity column.
authors.affiliation: a State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University , Nanjing , China.
authors.forename: Ming
authors.initials: M
authors.lastname: Shao
authors.forename: Zheng-Xin
authors.initials: ZX
authors.lastname: Peng
authors.forename: Chen-Yang
authors.initials: CY
authors.lastname: Shi
authors.forename: Rui
authors.initials: R
authors.lastname: Tang
authors.forename: Lawali Mamane
authors.initials: LM
authors.lastname: Manzo
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
mesh_terms: D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C068126:CD71 antigen; D011990:Receptors, Transferrin; D011994:Recombinant Proteins
keywords: human transferrin receptor; prokaryotic expression; protein purification; size exclusion chromatography