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Published on: February 5, 2015
[Cloning and expression of rat alpha-fetoprotein cDNA in Escherichia coli]
1Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, Japan.
Insights
Researchers cloned rat alpha-fetoprotein (AFP) cDNA and expressed it in E. coli. This recombinant AFP, useful for diagnostics, was purified and characterized, revealing it lacks the initial 53 amino acids of the precursor protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Alpha-fetoprotein (AFP) is a key serum protein synthesized during fetal development, primarily by the mammalian fetal liver and yolk sac.
- Reappearance of AFP in adult serum can indicate specific cancers like hepatoma or yolk sac tumors, as these tumors produce AFP.
- Understanding AFP's structure and production is crucial for diagnostic and research applications.
Purpose of the Study:
- To clone the complete coding region of rat AFP cDNA.
- To express and characterize recombinant rat AFP in E. coli.
- To facilitate further research and diagnostic development involving AFP.
Main Methods:
- Cloning of rat AFP cDNA.
- Expression of recombinant AFP in E. coli using an expression vector.
- Purification of recombinant AFP via immunoaffinity chromatography and SDS-PAGE.
- Analysis of nucleotide and amino acid sequences.
Main Results:
- Rat AFP was identified as being synthesized as a precursor with a 24-amino acid signal peptide and a 587-amino acid mature protein.
- Recombinant AFP produced in E. coli was immunologically reactive and had a molecular weight of 65,000.
- The purified recombinant AFP lacked the N-terminal 53 amino acid residues of the pre-AFP precursor.
Conclusions:
- The successful cloning and expression of rat AFP in E. coli provide a valuable tool for biochemical and immunological studies.
- The characterization of recombinant AFP confirms its identity and provides insights into its processing.
- This work supports the development of diagnostic tools and further research into AFP's biological roles.
Abstract:
AFP is a major serum protein during ontogeny and is synthesized mainly by the mammalian fetal liver and yolk sac. Its synthesis ceases early in postnatal life and its reappearance in the serum in adult is a sign of hepatoma or yolk sac tumor, since these tumors produce AFP. This paper describes the cloning of rat AFP cDNA spanning complete coding region, its expression in E. coli and characterization of this recombinant AFP. The determination of the nucleotide sequence and cell-free translation of purified AFPmRNA suggested that rat AFP was synthesized as a precursor with a signal peptide of 24 amino acids followed by mature AFP of 587 amino acids. An expression vector was constructed with the cDNA and the introduction of the plasmid into E. coli resulted in the production of immunologically reactive AFP with a molecular weight of 65,000. The recombinant AFP was highly purified by immunoaffinity chromatography followed by SDS-PAGE. Analysis of the amino acids sequence indicated that the product was AFP lacking N-terminal 53 amino acid residues of preAFP.
