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Human growth hormone: complementary DNA cloning and expression in bacteria.
Science (New York, N.Y.)
|August 10, 1979
Summary
Researchers cloned complementary DNA for human growth hormone (hGH) messenger RNA, revealing its prehormone sequence and signal peptide. This work supports evolutionary gene duplication and demonstrates high-level production of a stable, functional hGH fusion protein in E. coli.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Human growth hormone (hGH) plays a crucial role in growth and metabolism.
- Understanding the genetic basis of hGH is essential for studying its regulation and potential therapeutic applications.
- Previous studies on growth hormone sequences in different species have suggested evolutionary relationships.
Purpose of the Study:
- To clone and characterize the complementary DNA (cDNA) sequence of human growth hormone messenger RNA (mRNA).
- To predict the signal peptide sequence of human growth hormone.
- To investigate the evolutionary relationship between human growth hormone and related genes.
- To develop a method for high-level expression of human growth hormone in Escherichia coli.
Main Methods:
- Cloning of human growth hormone mRNA-derived cDNA.
- Nucleotide sequencing of the cloned cDNA.
- Bioinformatic analysis to predict protein sequences and evolutionary relationships.
- Gene fusion of human growth hormone sequences with the trp D gene of E. coli.
- Expression of the fusion gene in E. coli under the control of the trp operon.
Main Results:
- The cloned cDNA contains the 5' untranslated region (29 nucleotides), the coding sequence for the prehormone (651 nucleotides), and the 3' untranslated region (108 nucleotides).
- The signal peptide sequence of human growth hormone was predicted.
- Comparative sequence analysis supported the hypothesis of gene duplication evolution for hGH, rat growth hormone, and human chorionic somatomammotropin.
- A fusion protein, comprising 70% human growth hormone sequence, was synthesized at high levels (approx. 3% of bacterial protein) in E. coli.
- The fusion protein demonstrated specific reactivity with anti-hGH antibodies and was stable within E. coli.
Conclusions:
- The study successfully cloned and sequenced the full-length cDNA for human growth hormone.
- The predicted signal peptide sequence provides new insights into hGH processing.
- The findings strengthen the evolutionary link between growth hormone and related hormone genes.
- A robust system for producing a biologically relevant human growth hormone fusion protein in E. coli was established, with potential applications in research and biotechnology.