Production of Humulin by Recombinant E
-
Upload
bsreegiriprasad -
Category
Documents
-
view
47 -
download
0
Transcript of Production of Humulin by Recombinant E
![Page 1: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/1.jpg)
Production of Humulin by recombinant E. coliI.
![Page 2: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/2.jpg)
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.
![Page 3: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/3.jpg)
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.
![Page 4: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/4.jpg)
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.
![Page 5: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/5.jpg)
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.
![Page 6: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/6.jpg)
Inserting the gene into a vector (The plasmid).
![Page 7: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/7.jpg)
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.
![Page 8: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/8.jpg)
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.
![Page 9: Production of Humulin by Recombinant E](https://reader031.fdocuments.us/reader031/viewer/2022032721/55cf9953550346d0339ccbca/html5/thumbnails/9.jpg)
Try to answer this question.