Cloning the cDNA for horse growth hormone and expression in Escherichia coli.

The research paper examines the genetic cloning of the complementary DNA (cDNA) for the growth hormone in horses, followed by its expression in the bacteria Escherichia Coli. The study utilized a novel strategy to acquire the full cDNA sequence, which gets expressed in an expression vector and is analyzed subsequently.

Cloning and Sequencing of Horse Growth Hormone cDNA

• The researchers have cloned and sequenced a 514 base pair cDNA transcript that codes for horse growth hormone (GH), covering about 78% of the total.
• The derived amino acid sequence matched what had easily gotten from previous protein sequencing research.
• It also provided new sequence data for a critical component, the signal peptide.

Reconstruction of the Missing cDNA Fragment

• The 22% cDNA fragment that the initial clone didn’t cover was reconstructed using synthetic DNA strings, oligonucleotides, and site-directed mutagenesis.
• Mutagenesis, a technique used to modify the DNA, was precise and specific (site-specific) to ensure accuracy.

Use of an Expression Vector and Expression in E. Coli

• The complete cDNA sequence was then inserted into the vector PIN-III-lppp-5, designed to allow expression in the bacterium E. coli.
• This vector made use of a bacterial signal peptide to secret the expressed product into the periplasmic space of E. coli, the region between the outer and inner membranes of the bacteria.

Analysis and Performance of the Expressed GH

• The researchers utilized Western blot analysis, a routine molecular biology technique, on both cell lysates and periplasmic fractions prepared from cells harboring this construct. They found substantial amounts of immunoreactive GH.
• This analysis indicated that the bacterial signal peptide was successfully removed from the fusion protein during secretion.
• The GH extracted from the periplasm through osmotic shock was active in a heterologous radioimmunoassay and a horse liver radioreceptor assay, which means it functioned as expected.

Limited Secretion of Horse GH in E. Coli

• The research also highlighted a possible limitation. Even though horse GH was successfully expressed in E. coli, it seems there was a constraint on the secretion rate of horse GH.
• This potential block might have involved an issue regarding translocation across the cytoplasmic membrane and unfortunately limited higher levels of GH expression.