Cloning and expression of Human insulin gene Diabetes

Cloning and expression of Human insulin gene

Diabetes and the role of insulin Diabetes mellitus • Characterized by the excretion of large amount of glucose in urine. • Resulted from the body’s inability to make sufficient insulin. Insulin • A hormone excreted from the β cells in the pancreas, and involved in the regulation of blood sugar • At high level of blood glucose the insulin is secreted into the bloodstream, circulates throughout the body, and signals the liver and muscle cells to remove the excess glucose.

Diabetes and the role of insulin • Insulin recognizes specific insulin receptors on particular cells and initiates a cascade of reactions leading to glucose uptake • In absence of glucose uptake, individual cell will starve even plenty of glucose is available • Starving cells use fats as a primary source of energy leading to synthesis of ketone bodies • The ketone bodies and the glucose (high concentration) excreted in urine with huge amount of water.

Factors led to the development of human insulin production by bacteria • The steady increase in the incidence of diabetes • Allergic reactions due to animal insulin in some patients • Bacterial cells are easy to grow in mass quantity to provides a ready source for product

The first human gene product manufactured by recombinant DNA technology was human insulin, called Humulin, which was licensed for therapeutic use in 1982 by the U. S. Food and Drug Administration (FDA), the government agency responsible for regulating the safety of food and drug products and medical devices. In 1977, scientists at Genentech, the San Francisco biotechnology company cofounded in 1976 by Herbert Boyer and Robert Swanson isolated and cloned the gene for insulin and expressed it in bacterial cells. Genentech, short for “genetic engineering technology, ” is also generally regarded as the world’s first biotechnology company.

• Steps of insulin production from genetically engineered bacteria 1. Obtain the gene for insulin from human DNA • Insert the gene into bacterial cells (restriction endonuclease, vector, transformation) • Select the cells that have the desired gene • Induce the bacterial cells to express the inserted gene to produce insulin • Collect and purify the insulin

Obtaining the insulin gene • Finding the insulin gene in the DNA (finding needle in haystack • Isolation of m. RNA encoding insulin from pancreatic β cells • m. RNA is more abundant in β cells than the insulin gene • Eukaryotic m. RNA contains a poly A tail attached to one end of each RNA • This poly A tail can be used to isolate the m. RNA from other nucleic acids after breaking the β cell

Conversion of m. RNA to a complementary strand c. DNA by reverse transcriptase enzyme

Problem associated with obtaining the insulin from m. RNA • It is synthesized by the β cells of the pancreas as preproinsulin (inactive) • It is stored as proinsulin (inactive) and not released until it is needed by the body • Preproinsulin includes the signals necessary to direct the protein to storage. As it is being stored, the part of the protein responsible for signaling storage is specifically removed, leading to formation of proinsulin. • Proinsulin is the form that is stored.

• Active insulin is formed only when the body in need for insulin then proinsulin is activated by two specific cuts • The active form of insulin is actually two separate protein chains (A & B) held together by bonds

Clusters of cells embedded in the pancreas synthesize a precursor polypeptide known as preproinsulin. As this polypeptide is secreted from the cell, amino acids are cleaved from the end and the middle of the chain. These cleavages produce the mature insulin molecule, which contains two polypeptide chains (the A and B chains) joined by disulfide bonds. The A subunit contains 21 amino acids, and the B subunit contains 30. In the original bioengineering process, synthetic genes that encode the A and B subunits were constructed by oligonucleotide synthesis (63 nucleotides for the A polypeptide and 90 nucleotides for the B polypeptide).

Each synthetic oligonucleotide was inserted into a separate vector, adjacent to the lac. Z gene encoding the bacterial form of the enzyme bgalactosidase. When transferred to a bacterial host, the lac. Z gene and the adjacent synthetic oligonucleotide were transcribed and translated as a unit. The product is a fusion protein—that is, a hybrid protein consisting of the amino acid sequence for b-galactosidase attached to the amino acid sequence for one of the insulin subunits The fusion proteins were purified from bacterial extracts and treated with cyanogen bromide, that cleaves the fusion protein from the bgalactosidase. When the fusion products were mixed, the two insulin subunits spontaneously united, forming an intact, active insulin molecule.

Is there a way to get the cells to make active insulin instead of inactive preproinsulin? • If each of two chains of the active insulin could be made separately and then properly assembled with the other, active insulin could be produced • This is the way recombinant insulin was finally produced. • A gene encoding information for one insulin chain was cloned into bacterial cells. The chain was expressed and purified from these cells • The information for the other chain was cloned separately, ultimately providing purified second chains

• The two strands were mixed outside the cells and bound to each other in proper fashion to form the active insulin molecule • Thus, cloned insulin was really produced as a mixture of two separately cloned products.

• Because the sequence of amino acids that comprise the two chains was known, this information was used in conjunction with the genetic code to work backward i. e. protein to DNA • An advance technique in which fully automated computer process (DNA synthesizer) is finally made it possible to complete the cloning of insulin • To obtain insulin genes, the nucleotides sequences of the two chains of the insulin protein are assembled by using DNA synthesizer rather than the m. RNA reverse transcription. •

Inserting the insulin gene into bacterial cells • The synthesized DNA segments are incorporated within vector eg plasmid • The synthesized DNAs do not contain the same recognition site to make the same sticky ends so small artificial pieces of DNA (linkers) that contain the same restriction enzyme recognition sequence as the plasmid are added • The linkers are attached to the synthesized DNAs by ligase enzyme then digested with the same restriction enzymes to give the same sticky ends. • The vector is then introduced into bacterial cells (competent) by transformation

Inducing the expression of insulin in bacterial cells. • Insulin may be degraded by bacterial cells. • Once produced by bacterial cells, insulin may be degraded by protease enzyme • Use genetically engineered bacteria deficient of protease gene.

Purifying the product (downstream processing)