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A Convenient Method For hTfR1 Inclusion Body Purification
- Shao, Ming, Peng, Zheng-xin, Shi, Chen-yang, Tang, Rui, Manzo, Lawali Mamane, Liu, Yu
- Preparative biochemistry & biotechnology 2015 v.45 no.8 pp. 743-753
- Escherichia coli, Western blotting, antibodies, blood-brain barrier, drugs, endocytosis, gel chromatography, genes, human cell lines, humans, nickel, recombinant proteins, reverse transcriptase polymerase chain reaction, tissues, transferrin, transmembrane proteins
- 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.