How to make a protein in the laboratory: sH proteins
Nature took its time, like some millions of years, and ended up with a complicated and beautiful system that works well to make proteins. When it doesn’t, the consequences can be devastating, such as when DNA that codes for an important protein mutates because of bad luck or environmental factors.
How does the system work? It’s complicated
Figure. Protein synthesis, or gene expression, is a two-stage process. In the first (transcription) a mRNA molecule is produced that echoes the DNA sequence of the gene being expressed. In the second stage, translation, a chain of amino acids is formed at the ribosome, following the instructions encoded in the mRNA.
The amino acid sequence of proteins is determined by the nucleotide sequence in the coding regions of DNA. The DNA that codes for proteins is transcribed into a complementary messenger RNA (mRNA) molecule. Each amino acid is coded for by a sequence of three nucleotides in mRNA.
Protein synthesis occurs in the cytosol on ribosomes. Ribosomes are cytoplasmic complexes made up of proteins and ribosomal RNA (rRNA). After leaving the nucleus, mRNA molecules become associated with ribosomes. The mRNA molecule serves as a template, and the ribosome provides the structural framework and catalytic activity for protein synthesis.
The mRNA codons are recognized by transfer RNA (tRNA) anticodons. Transfer RNA molecules a single-stranded RNAs that fold (due to intramolecular base pairing) into a cloverleaf structure. Codon recognition is the result of base pairing between the nucleotides of the mRNA strand and a three-nucleotide region (anticodon) on one “arm” of the tRNA molecule. Because a given tRNA must recognize both a specific mRNA codon and a specific amino acid, tRNA molecules can be thought of as adapters linking two dissimilar entities.
The 3′ end of the tRNA molecule is the point of attachment for its amino acid. Polypeptide synthesis is initiated at the amino-terminal end of the protein and requires an AUG codon. Polypeptide elongation involves the formation of peptide bonds between amino acids on adjacent tRNAs. Termination of polypeptide synthesis requires one of three termination codons and one of three termination factors.
How does a biochemist make a protein in large quantities?
We ask a bacterium like Escherichia coli, or a plant cell, or a fungal cell, to do the job. It will use the same machinery it was using before to make its own proteins, but now it will be working for us. How? We introduce a piece of DNA that codes for “our” protein, and we direct it to stop everything else and get going.
Figure. To clone a stretch of DNA into a vector, restriction enzymes are used to cut the DNA of interest and and open up the vector. The DNA is added to the vector by mixing the two together in the presence of the enzyme DNA ligase. Nowadays, we skip the first step and start with a synthetic piece of DNA coding for the protein but optimized for E. coli. This way, we get a “sH protein” in the INCI nomenclature. The vector will contain a piece of DNA coding for the human protein but avoiding the use of human DNA.
The result is the same: we get cells cultured in a flask to produce large amounts of the protein we want. When we have enough, we break the cells and recover the protein we want. Using sophisticated biochemical methods, we purify the desired protein to homogeneity, and voilà, there it is. The advantage? What used to require tons of source material (liver? urine? whatever) is now in our hands after a few days of cell culture. That, plus many decades of scientific research, and many Nobel prizes.
At Skin Actives Proteins, we use E. coli, a bacterium, a fast and reliable operator. We have many decades of expertise in optimizing the synthesis and purification of the proteins we make, resulting in a highly pure, very active protein identical to the one our body makes. Because of our uncommon background, we can sell it at a fraction of the price others charge, making it practical to use in skin care products at a concentration that delivers excellent, visible results.
Hannah