Cloning Human Insulin Gene

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Before the advent of biotechnology, the insulin used for the treatment of type I (insulin-dependent) diabetes mellitus was obtained from extracting the hormone from porcine or bovine pancreatic tissues. In the early eighties, human insulin produced by recombinant technology entered the pharmaceutical market.

In one of the approaches (Geoddel et al. 1979. Proc. Natl. Acad. Sci. USA 76, 106-110), the sequences for the A and B chains were synthesized chemically and inserted separately downstream of the P-galactosidase structural gene controlled by the X lac promoter. The construction was such that the insulin chains would be made as fusion proteins joined by a methionine to the end of the p-galactosidase protein (see Section 9.1.1). The expression vector also contained an AmpR marker. Transformants were screened by plating on a culture medium containing X-gal and ampicillin. Insulin A chain and B chain transformants were grown to harvest the cells in large quantity. The cells were lysed and the insulin A chain and B chain were purified separately. Because the insulin A gene was fused to the P-galactosidase gene, therefore the insulin protein produced was a P-galactosidase-insulin hybrid.

Amp1

Amp1

J Expression

B chain

B chain

Expression

J Expression

13-931 \ A chain CZZ2ZZZZZZ2

I CNBr treatment

I Isolation I Purification

Expression

B chain i

CNBr treatment

| Isolation I Purification

Combined Reduced-reoxidized

I I Active insulin

Fig. 16.2. Strategy for cloning and production of human insulin.

This hybrid protein was treated with cyanogen bromide to cleave off the insulin chain at the methionine. Likewise, insulin B chain also underwent the same treatment. The purified insulin A and B chains were mixed and subjected to reduction-reoxidation to insure correct joining of the disulfide bonds (Fig. 16.1). An alternative procedure involves the cloning of human pro-insulin (A-C-B sequence) into bacterial cells. The C-peptide of the expressed protein was enzymatically cleaved to yield the active A-B form.

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Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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