Production of Humulin by recombinant E. coliI.

Finding the insulin gene• Method 1: Using reverse

transcriptase to synthesise cDNA.

– Extract and purify mRNA from Beta cells from the islets of Langerhans.

– Incubate mRNA with reverse transcriptase and activated deoxyribonucleotides.

– Remove original mRNA strand using RNAase or alkali to produce a single stranded cDNA copy.

– Complete the double stranded DNA molecule using DNA polymerase and activated deoxyribonucleotides.

– Sticky ends are added.

Finding the insulin gene• Method 2: Using chemical

synthesis of DNA molecule using the base sequence gleaned from the primary structure of the two polypeptides.

– Synthesise the 63 and 90 polynucleotide chains coding for chain A and B of insulin.

– Add a stop codon at the 3’ end.– At the 5’ end add methionine

anticodon.– Sticky ends are added.

Finding the insulin gene

• Method 3: Make a genomic library of the DNA.

• Extract all DNA from beta cell and perform a ‘partial digest’. This involves using a restriction endonuclease (e.g. SAU 1A) and cutting the genetic information into smaller pieces.

• These DNA fragments are inserted into a vector and cloned to form a genomic library.

• The library can then be screened using a radioactive probe for the insulin genes and the genes cut out using restriction endonucleases, producing sticky ends.

Inserting the gene into a vector (The plasmid).

• Plasmid is a small circular piece of DNA.• They can contain genes which confer antibiotic

resistance.• To insert gene the plasmid is cut open using a

restriction endonuclease, leaving a short length of unpaired bases at each end (sticky ends).

• The DNA is inserted using the sticky ends complementary to those on the plasmid and the phosphodiester bond formed using an enzyme called DNA ligase.

• This is called recombinant DNA.

Inserting the gene into a vector (The plasmid).

Inserting the vector into the required organism (E. coli).

• The recombinant plasmid is inserted into the bacteria by the process of transformation.

• The recombinant bacteria are sorted by growing them in the presence of an antibiotic. The bacteria which survive are the ones which have taken up the plasmid.

• They are said to be transformed.

Culturing recombinant E. coli and insulin synthesis.

• These recombinant bacteria are then produced in a monoculture and the two insulin chains are synthesised by the recombinant bacteria.

• The two polypeptides accumulate in the fermentation liquor and are extracted and purified.

• Insulin is finally formed when the two polypeptide chains are joined using disulphide bonds.

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