This document discusses the production of human insulin using gene technology. It describes how the human insulin gene is isolated and inserted into bacterial plasmids using restriction enzymes. The recombinant plasmids are then transformed into bacteria which are selected and grown to produce human insulin. Producing insulin through gene technology eliminates issues with immune responses to animal insulins and is more cost effective than extracting insulin from animals.
This document discusses the production of human insulin using gene technology. It describes how the human insulin gene is isolated and inserted into bacterial plasmids using restriction enzymes. The recombinant plasmids are then transformed into bacteria which are selected and grown to produce human insulin. Producing insulin through gene technology eliminates issues with immune responses to animal insulins and is more cost effective than extracting insulin from animals.
This document discusses the production of human insulin using gene technology. It describes how the human insulin gene is isolated and inserted into bacterial plasmids using restriction enzymes. The recombinant plasmids are then transformed into bacteria which are selected and grown to produce human insulin. Producing insulin through gene technology eliminates issues with immune responses to animal insulins and is more cost effective than extracting insulin from animals.
This document discusses the production of human insulin using gene technology. It describes how the human insulin gene is isolated and inserted into bacterial plasmids using restriction enzymes. The recombinant plasmids are then transformed into bacteria which are selected and grown to produce human insulin. Producing insulin through gene technology eliminates issues with immune responses to animal insulins and is more cost effective than extracting insulin from animals.
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0 GENE TECHNOLOGY 1.1 INSULIN PRODUCTION Objectives
By the end of the subtopic learners should be able to:
1. Define gene technology
2. Outline the synthesis of human insulin by bacteria 3. Explain the advantages of treating diabetics with human insulin produced by gene technology.
Gene technology involves the artificial manipulation, modification, and recombination of
DNA or other nucleic acid molecules in order to modify the genetic makeup of organisms. DNA molecules from two or more sources are combined either within cells or in vitro. There are then inserted into host organisms in which they are able to propagate. The method creates new combinations of genes and is referred to as genetic engineering. Genetic engineering in bacteria can be broken into five stages (fig.1.1.1).
Fig.1.1.1: Stages of genetic engineering.
Some of the applications of genetic engineering include:
o Basic research on gene structure and function
o Production of useful proteins by novel methods for example insulin o Generation of transgenic plants and animals. o Medical diagnosis and treatment o Genome analysis by DNA sequencing
Insulin production
Insulin is a hormone that is produced and secreted by the beta cells of the pancreas' Islets of Langerhans. It is a simple protein which consists of two polypeptides. The hormone has a total of 51 amino acids, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain. Insulin is responsible for controlling blood glucose levels and its shortage causes a condition referred to as diabetes. Bacteria are used as hosts in insulin production because of their ability to readily take up plasmids and produce proteins carried by the plasmid.
Steps for insulin production
Insulin can be produced using bacteria (E.coli) through Recombinant DNA technology. Recombinant DNA technology involves joining together DNA from different species. Several steps are involved in insulin production as outlined below.
1. Extraction o The plasmid DNA is isolated from bacteria. o A copy of the human insulin gene is obtained. o This is done by isolating mRNA from the human pancreas.
2. Digestion by restriction enzymes
o Restriction enzymes are used to cut the plasmid DNA. o Plasmid DNA contains specific sequences which are known as restriction sites to allow cleavage of specific sites by restriction enzymes. o Restriction enzymes cut DNA at specific points by recognising specific sequences on the DNA molecule. o They are naturally found in bacteria and they protect bacteria against viruses. o Restriction enzymes can leave either open stick ends (length of unpaired bases) or blunt ends. o The same restriction enzymes (restriction endonucleases) are also used to cut insulin DNA. o However, they do not digest their host DNA because bacterial DNA is methylated.
Fig.1.1.2: Restriction digestion of the plasmid and the insulin gene
3. Insertion o The isolated insulin DNA is then inserted into a plasmid vector. o The cut plasmids are mixed with the insulin gene and DNA ligase enzymes are added. o DNA ligase enzymes are enzymes that are used to join together plasmid DNA with the insulin DNA to form recombinant DNA molecule Fig.1.1.3: Diagrammatic representation of the insertion step.
4. Transformation o The recombinant molecule is then inserted back into the bacterium which will act as a host. o The process whereby bacteria take up recombinant molecules is referred to as transformation. o The cells which have taken up the plasmid are called transformants. o Heat shock is usually introduced to induce transformation.
5. Selection o The transformed cells are identified and selected basing on marker genes. o A marker gene is a gene that allows genetically modified cells to be readily selected. o An example is the gene that encodes antibiotic resistance. o Antibiotic resistance will act as a selectable marker. o These selectable markers are found on plasmids. o The bacterial host cell will divide and produce copies of the plasmid carrying the insulin gene (fig 1.1.4).
6. Extraction and purification
o Insulin is then extracted from the bacterial culture. o It is then purified. Fig.1.1.4: Summary of the steps in the production of insulin
Advantages of using insulin to treat diabetes`
Before bacteria was used to produce human insulin diabetes were injected with insulin isolated from pigs or cattle. However, pig or cattle insulin is not identical to human insulin and there were cases of immune response. There are minor differences in the amino acid composition between cow or pig insulin and human insulin (fig.1.1.5). Fig.1.1.5: Differences between pig and human insulin.
Insulin produced using bacteria is chemically similar to human insulin and there are less chances of immune response. It can also be easily recognised by human insulin receptors on human cell surface membranes hence duration of response is shorter. Insulin produced by gene technology eliminates the problems related to the development of tolerance to cow or pig insulin. Ethical issues raised by use of cow or pig insulin are eliminated when insulin is produced through gene technology. For instance, religious objections of pig insulin by Muslims and Christians or objections of both pig and cattle insulin by vegetarians. Extraction of insulin from pancreas of cows or pigs is expensive as compared to production of insulin through gene technology. Production of insulin through gene technology also eliminates problems related to animal rights issues as there is no need to slaughter any animal.
