Expression of Ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: Its Purification and Immunohistochemical Detection Using Monoclonal Antibody

• Published in: Protein expression and purification Vol. 24; no. 2; pp. 292 - 301
• Main Authors: Chiu, Chi-Chien, Christopher John, Joseph Abraham, Hseu, Tzong-Hsiung, Chang, Chi-Yao
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-03-2002
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal; ayu; Base Sequence; Blotting, Western; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Escherichia coli; fish; Fishes; Immunohistochemistry; Mice; Mice, Inbred BALB C; Molecular Sequence Data; monoclonal antibody; Pit-1; Pituitary Gland - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Transcription Factor Pit-1; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism; immunohistochemistry; ayu; monoclonal antibody; Pit-1; fish
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2001.1558

The pituitary-specific transcription factor Pit-1 belongs to the family of POU-domain proteins and is known to play an important role in the differentiation of pituitary cells. Here we report the complete nucleotide sequence of cDNA encoding Pit-1 from the brackish water fish, ayu (Plecoglossus altivelis). Nucleotide sequence analysis of 1910 bp of ayu Pit-1 cDNA revealed an open reading frame of 1074 bp that encodes a protein of 358 amino acids containing a POU-specific domain, POU homeodomain, and an STA (Ser/Thr-rich activation) transactivation domain. We inserted the coding region of Pit-1 cDNA, obtained by PCR, into a pET-20b(+) plasmid to produce recombinant Pit-1 in Escherichia coli BL21 (DE3) pLysS cells. Upon induction with isopropyl β-d-thiogalactopyranoside, Pit-1 was expressed and accumulated as inclusion bodies in E. coli. The protein was then purified in one step by affinity chromatography on a nickel-nitrilotriacetic acid agarose column under denaturing conditions. This method yielded 0.7 mg of highly pure and stable protein per 200 ml of bacterial culture. A band of 40 kDa, resolved as recombinant ayu Pit-1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, agrees well with the molecular mass calculated from the translated cDNA sequence. The purified recombinant Pit-1 was confirmed in vitro through Western blot analysis, using its monoclonal antibody. This monoclonal antibody detected Pit-1 in the nuclei of ayu developing pituitary by immunohistochemical reaction. It serves as a good reagent for the detection of ayu Pit-1 in situ.