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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.