- Cell fractionation by conventional chromatographic methods to yield about a microgram of protein.
- The protein is analyzed to yield the identity of the first 30 amino acids - its N-terminal amino acid sequence.
- The Genetic Code is used to predict nucleotide sequences corresponding to this amino acid sequence.
- DNA fragments complementary to these sequences (oligomers of 15-20 bases) are chemically synthesized.
- The DNA fragments are then hybridized (base-paired) with total cellular mRNA.
- Long cDNA segments are produced from mRNAs complementary to the DNA fragments using the enzyme reverse transcriptase.
- Large amounts of this cDNA is obtained by cloning into plasmids (amplification).
- Selection of the right clone (several steps).
- Finally, the cloned cDNA is incorporated into an expression vector or plasmid an_d transferred into bacterial or yeast cells.
- This is the starting point for scaled-up production of large amounts of the protein.