The Master Mix for Insulin: Raw Materials

Raw materials are the most basic components to produce a product. In this section, some of the typical crude raw materials that lead to the industrial production of synthetic insulin will be introduced to you.

- Raw materials used for preparation of recombinant plasmid

(1) Desired Gene

The human insulin gene isolated from the human DNA. However, often, mRNA encodes for insulin is used because finding one gene in the human genome that consists of some 70 000 genes can be considered a mammoth task. Because the B cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene as this is much simpler.

(2) Vector

Plasmid is the most preferred vector if bacterial host cells are used. The general features of plasmid in insulin production include:

1. It usually replicates independently of bacterial chromosome to produce higher yield of products
2. Permits the reproduction of a foreign DNA by using the bacterial replicating system.
3. Usually contains selective markers to eliminate undesired cells
4. Suitable to accommodate the size of the insulin gene

(3) Specific enzymes

Reverse transcriptase - synthesizes cDNA from the mRNA template that is requiredfor the insertion into the vector

Specific restriction enzymes – to cut DNA at specific sites, such as on the vector for the insertion of the desired insulin gene.

Specific ligases – to join DNA fragments together after the insulin gene has been inserted into the vector so that the gene can be expressed in a host cell.

- Raw Materials used for the fermentation process

(1) The host organism

The bacteria Escherichia coli is commonly used as the host organism to produce the synthetic insulin. However in the 1990s, the insulin production method was improved by using the yeast Saccharomyces cerevisiae instead of E. coli.

This is due to the fact that bacterial cells cannot do post-translational modification. After translation, post-translational conversion to insulin was carried out chemically. By contrast, yeast, as a eukaryote, is capable of post-translational modification, so this simplifies the production of human insulin. (E. coli is used for this website.)

E. coli

Saccharomyces cerevisiae

(2) Media

Luria-Bertani (LB) Medium is used for the production. It is used as both seed-culture and the fermentation media, especially for the cultivation of Escherichia coli. The recipe of LB contains: 1) Bacto-tryptone, 2) Yeast extract, 3) Sodium chloride, 4) dH₂O and 5) pH of 7.5.

Other than the LB components, 2 more components – ampicillin and lactose are present in the media. These two unique components convert the medium into an enrichment liquid culture such that only the desired bacteria that contain the human insulin gene can grow in this type of media. On how it works will be further described in the process description section.