A convenient method for hTfR1 inclusion body purification

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Human transferrin receptor (hTfR1) is highly expressed in the blood-brain barrier and tumors.
• hTfR1 is a type II homodimeric transmembrane protein with a specific extracellular domain structure.
• Targeting hTfR1 is crucial for drug delivery across the blood-brain barrier via receptor-mediated endocytosis.

Purpose of the Study:

• To develop a method for expressing the nonligand binding domain of hTfR1 in Escherichia coli.
• To prepare hTfR1 inclusion bodies for antibody production.
• To establish a protocol for utilizing hTfR1 in drug delivery systems.

Main Methods:

• Obtained the TfR1 gene from HepG2 cells using reverse-transcription polymerase chain reaction (RT-PCR).
• Inserted the TfR1 gene into the pET 32a(c+) vector for expression in E. coli BL21 Transetta (DE3).
• Purified the expressed protein using size-exclusion chromatography and confirmed identity via Western blot.

Main Results:

• Successfully expressed the recombinant hTfR1 protein as an inclusion body with high purity in E. coli.
• Size-exclusion chromatography further enhanced the purity of the inclusion body.
• Western blot analysis confirmed the identity of the recombinant protein as hTfR1.

Conclusions:

• A convenient protocol for preparing hTfR1 inclusion bodies was established.
• The developed method provides a foundation for generating hTfR1 antibodies for therapeutic applications.
• This approach facilitates drug delivery across the blood-brain barrier by targeting hTfR1.