1. Steps Involved in Human Insulin Production by
Recombinant DNA Technology/ Genetic Engineering
Dr. Shamim A. Qureshi, PhD
Professor
Department of Biochemistry,
University of Karachi, Pakistan
2. Human Insulin
Insulin is a hormone produced by β-cells of islets of Langerhans of
pancreas.
Structure of Human Insulin:
• Insulin (51 amino acids; MW 5808 Da.) is protein hormone.
• Consists of two polypeptide chains: chain A (21 amino acids) and chain B (30
amino acids).
• Both chains (A and B) are connected together by two inter-chain disulfide bonds:
i. Between Cysteine (Cys) residues present at 7 position of both chain A and B
ii. Between Cys residues present at position 20 of chain A and at position 19 of
chain B
An intra-chain disulfide bond is also present in A chain btw Cys (6) and Cys (11).
• The synthesis of each polypeptide chain (A & B) of insulin is under the control of
two separate genes on DNA.
4. Steps Involved in Human Insulin Production in E.coli by
Recombinant DNA Technology/ Genetic Engineering
• DNA sequence of insulin genes for both chains (A and B) can be cut with help of RE from
human chromosome (s) OR chemically synthesized (Hakura et al, 1977) and
inserted/ligated separately into the RS of two pBR322 plasmids (expression vector).
• pBR322 has promoter sequence (binding site for RNA polymerase), followed by lac z
gene /β-gal gene (encodes β-galactosidase enzyme) and restriction site (cut by
restriction endonucleases such as EcoR1, BamH1). It has also a marker gene antibiotic
resistant (ampicillin resistant; AmpR) gene (used to select transform and non-transform
bacterial cells).
• The genes (A & B chains) are separately inserted/ligated (with the help of DNA
ligase) into the restriction site adjacent to the lac z/ β-gal gene of two pBR322
plasmids to form recombinant plasmids.
5.
6. • The recombinant plasmids then separately transferred into separate E. coli (host cells) and
cultured them in nutrient broths (liquid mediums in conical flasks/fermenters) separately.
• In liquid mediums, not only transformed bacterial cells replicated but also produced pro-
insulin/fusion protein as β-galactosidase (β-gal)-A chain protein and β-gal-B chain protein
separately.
• These transformed bacterial cells then homogenized separately to rapture the cells and to
obtain βgal-chain A & B fusion proteins in supernatants.
• These supernatants not only contain fusion β-gal-chain A and B proteins but also contain
other proteins and contaminants.
• These supernatants then applied on top of the two separate columns of affinity
chromatography, where β-gal-A chain and β-gal-B chain fusion proteins are retained on
sepharose gel beads (stationary phase) having antibodies (as ligands) to which β-gal part of
fusion proteins has affinity to hybridize or bind with. Whereas other proteins and
contaminants come out of the columns with buffer (mobile phase). Then bound β-gal-A
chain and β-gal-B chain fusion proteins from gel beads get detached with the help of elution
buffer and eluted out of their respective columns and collected in test tubes/cuvettes
separately.
• Then both chains (A and B) get separated from β-galactosidase part by treating with
cyanogen bromide (CNBr).
• Finally, isolated A and B chains are then linked invitro to produce mature insulin (functional
insulin) by forming inter- and intra-chain disulfide bonds through sulphonating the cys
residues of chains A & B with sodium disulphonate and sodium sulphite.
