Recombinant Protein Synthesis Steps …

Proteins Are Any molecule that is involved in the maintenance and metabolic processes of living organisms. Biomolecules include carbohydrate, lipid, protein, water and nucleic acid.Proteins in living organisms are classified according to their biological roles i.e Enzymatic,hormonal,structural,receptors,contractile,defensive,strorage&transport.Humans have traditionally used animal and plant sources to obtain proteins (such as hormones and clotting factors) in order to treat different diseases and conditions. Many of these were extracted from human and animal corpses.

Protein-Based Drugs.

Prior to advances in biotechnology such as rDNA technology, the few protein drugs available were taken directly from human and animal corpses. For instance, the human growth hormone was taken from human corpses and insulin required to treat diabetes was collected from slaughtered pigs. These drugs were available in limited supply and they were expensive, given their sources. Biotechnology has boosted the production of protein-based drugs in a significant way i.e Recombinant DNA Technology

The Chemical makeup of DNA and RNA -

1.DNA – is made up of many DNA nucleotides (nucleotides are the building blocks of nucleic acids). Each DNA nucleotide is composed of deoxyribose (a pentose, or five carbon sugar),a phosphate molecule and one of four nitrogenous bases: Adenine, Thymine, Cytosine and Guanine. (A, T, C, G). The picture below shows the double stranded DNA structure and complementary bases of the DNA bases.  

2.RNA – is made up of many RNA nucleotides (nucleotides are the building blocks of Nucleic acids). Each RNA nucleotide is composed of ribose (a pentose, or five carbon sugar), a phosphate molecule and one of four nitrogenous bases Adenine, Uracil, Cytosine and Guanine. (A, U, C, G).

3.DNA and RNA Nucleotide DiagramsThe diagrams below show different diagrams of nucleotides, both RNA and DNA. Be able to recognize the sugar, the five sided figure in the first two diagrams and the five sided figure labeled Y in the third diagram, the phosphate (the circle with the P or Z inside) and the base (the six sided figure with the N in its structure, labeled W or X in the third diagram) The last diagram shows 4 nucleotides, showing bases, sugars and phospates

 

RNA & DNA

Recombinant DNA Technology

Genetic engineering,is a technique in which the gene that encodes for the required protein is transferred from one organism into another, which is capable of producing large amounts of the drug.Recombinant DNA - contains DNA from 2 different sources. Organisms that have had a foreign gene inserted into them are called transgenic organisms

Importance of Recombinant DNA technology

Objectives of DNA technology include,identifying,isolating,modifying &re expressing genes in other hosts or organism.

The introduction of rDNA technology or genetic engineering, has provided a large and cheaper source of protein-based drugs .Genetic engineering plays very important role not only in scientific research but also in diagnosis and treatment of disease.Genetic engineering produces proteins that offer advantages over proteins isolated from other biological sources like high purity,high specific activity,steady supply&batch to batch consistency

It permit scientists &clinicians to identify new gene&proteins they encode,to correct endogenous genetic defect&to manufacture large quantities of specific gene products such as hormones,vaccines &other biological agents of medical interest.

Use of Recombinant DNA research

some of the areas where Recombinant DNA will have an impact,Better Crops (drought & heat resistance)Recombinant Vaccines (ie. Hepatitis B) Production of clotting factors Production of insulin Production of recombinant pharmaceuticals Plants that produce their own insecticides Germ line and somatic gene therapyPrevention and cure of sickle cell anemia Prevention and cure of cystic fibrosis

.Different type of protein based drugs produced by recombinant

DNA technology Cytokines - these drugs regulate the immune system. That is, they are proteins that activate the immune system cells to carry out different immune functions. Hormones - protein drugs that regulate functions in the body. As drugs, these proteins can be used to elevate levels of certain hormones, such as estrogen during menopause or growth deficiency. They can also be used to treat certain diseases such as

diabetes or conditions such as infertility

Clotting factors. proteins that regulate the clotting of blood. These drugs are used to treat blood clotting disorders such as hemophilia. Vaccines - proteins that stimulate the immune system to produce specific antibodies used to prevent or treat diseases.

Monoclonal antibodies- proteins that mark a specific foreign material (such as cancer cells, disease-causing bacteria and viruses), for removal or destruction by other components of the immune system. These are also used as effective diagnostic tools for many specific genetic diseases and other conditions such as pregnancy

Genetic engineering has permitted

Identification of mutations:People may be tested for the presence of mutated proteins that may be involved in the progression of breast cancer, retino-blastoma, and neurofibromatosis.Diagnosis of affected and carrier states for hereditary diseases:Tests exist to determine if people are carriers of the cystic fibrosis gene, the Huntington’s disease gene, the Tay-Sachs disease gene, or the Duchenne muscular dystrophy gene.

Mapping of human genes on chromosomes:Scientists are able to link mutations and disease states to specific sites on chromosomes.Performing structure and function analyses on proteins:Researchers may now employ rational drug design to synthesize drug compounds that will be efficacious and selective in treating disease.

Transferring genes from one organism to another:People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and certain cancers may now benefit from the progress made in gene therapy.

Isolation and alteration of genes:Once gene modification becomes successful, alteration of genes to produce a more functional protein than the endogenous protein may become possible, opening up the route of gene therapy.

Isolation of large quantities of pure protein:Insulin, growth hormone, follicle-stimulating hormone, as well as other proteins, are now available as recombinant products. Physicians will no longer have to rely on biological products of low purity and specific activity from inconsistent batch preparations to treat their patients.

 Steps in Synthesizing a Recombinant Protein:

Recombinant Protein synthesis is a complicated process to illustrate and includes many steps but simply it can be expressed as under..Protein expression,transcription and translation,Protein purification (small- through large-scale),Protein characterization.Quality assurance .Specialized services—custom cell-based assays & custom antibodies, libraries, and cloningprotein expression.

. Recombinant technology begins with protein expression as following.

These are charts to describe the whole procedure illustrating recombinant protein production steps.

.isolation of a gene of interest by using restriction enzymes.insersion of gene of interest into a vector by using integration enzymes,ligases.( A vector is a piece of DNA that is capable of independent growth; commonly used vectors are bacterial plasmids and viral phages).

introduction or integration of The gene of interest (foreign DNA)into the plasmid or phage(and this is referred to as recombinant DNA){Plasmids are circular, double-strandedDNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells.They can replicate independently of the host cell.The size of plasmids ranges from a few kb to near 100 kb,Can hold up to 10 kb fragments,have an origin of replication,

antibiotic resistance genes as markers, and several unique restriction sites.After culture growth, the clone fragment can be recovered easily. The cells are lysed and the DNA is isolated and purified.A DNA fragment can be kept indefinitely if mixed with glycerol in a –70 degrees C freezer)}.cloning of vector is done(Before introducing the vector containing the foreign DNA into host cells to express

the protein) )(Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells.){Cloning Vectors.A vector is used to amplify a single molecule of DNA into many copies.

A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell.Most vectors are genetically engineered plasmids or

phages.

. There are also cosmid vectors, bacterial chromosomes, and yeast artificial chromosomes}.isolation of the vector having foreign DNA in large quantities.introduction of vector into the desired host cells such as mammalian, yeast, or special amounts.

bacterial cells..synthesis of foreign protein by host cells from the recombinant DNA..isolation of foreing or recombinant protein When the cells are grown in vast quantities.

purification of foreign protein in large amounts. it is important for saving the integrity and properties of the proteins. Basically, the purification is based on affinity chromatography. However, if a high purity grade or polishing step is required, we combine/associate additional techniques: ions exchange, gel filtration, hydrophobic interaction chromatography.After this other steps are done.