processing of recombinant proteins
- 1. Processing of recombinant proteins Extracellular and pericellular production of recombinant proteins Silpa Mohandas MSc Biotechnology
- 2. Recombinant proteins Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins, modify gene sequences and manufacture useful commercial products. The formation of recombinant protein is carried out in specialized vehicles known as vectors. Recombinant technology is the process involved in the formation of recombinant protein.
- 4. Recombinant proteins ■ Recombinant Proteins: Proteins are complex biomolecules that play various fundamental roles in living systems. They are made up of building blocks called amino acids that are attached to one another in chains. ■ Proteins occur in different types and serve functions like structural support, mediation of cell processes, the regulation of cells and tissues, and the expression of physical attributes like eye color, height, and much more. ■ The advent of modern technologies has undeniably brought so much improvement in the study of protein molecules. For instance one of the main goals of the biotechnological field is the production of recombinant proteins.
- 5. History of recombinant proteins ■ The idea of recombinant DNA and the method of producing it were made by a graduate student Peter Lobban of Stanford University. Along with his professor, Dale Kaiser, the two the published their hypotheses in the journal Enzymatic end-to-end- joining of DNA molecules (1973) and explained the method of separating and amplifying genes and inserting them to a new host cell. ■ In 1974, Stanford University applied for patent (with Stanley Cohen and Herbert Boyer as inventors) and was awarded in 1980. ■ New advancements in the technology arose such
- 6. Two methods of producing recombinant proteins ■ The process of producing recombinant proteins is highly dependent on the process of inserting the DNA segment to the host’s genome. Two methods are known to produce recombinant DNA namely : ■ Molecular cloning ■ Polymerase chain reaction
- 7. 1.Through Molecular Cloning ■ Molecular cloning is a process that is used to insert a recombinant DNA into a vector (vehicle used to transfer genetic material) that will replicate the segment inside the host organism. The DNA segment may be isolated from either a prokaryote or a eukaryote bearing the gene or protein of interest. ■ Following the isolation of the DNA segment is its cutting (with restriction enzymes) and inserting into the plasmid vector through ligation. ■ The resulting product is now called a recombinant plasmid and is now ready to be inserted into the host (which is in the case of the illustration below is the bacterium E. coli).
- 8. production of recombinant proteins
- 10. Through polymerase chain reaction ■ Also referred to as “Molecular Photocopying “, Polymerase Chain Reaction (PCR) is a method to quickly amplify segments of the DNA into million copies. Once amplified, the copies of the DNA segments produced can now be used in various laboratory experiments like mapping. Check out the History of Genetics and the year 1983 section for more discussion on PCR. ■ Unlike molecular cloning, PCR produces recombinant DNA without the need for vectors and host cells. ■ The templates DNA (DNA to be copied) is simply mixed with the forward and reverse primers, nucleotides, and DNA polymerase and undergo repeated cycles through PCR.
- 11. PCR ■ PCR, polymerase chain reaction, is an in- vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence. Requirement ■ DNA template ■ Primers ■ Enzyme ■ dNTPs ■ Mg2+ ■ buffers
- 12. ■ Comprises of 3 steps,
- 17. Insulin ■ Insulin is produced and secreted by beta cells of Langerhans pancreas, that regulates the use and storage of carbohydrates. Structure of Insulin ■ Chemically, insulin is a small and simple protein , consists of 51 amino acids, 30 construct 1 polypeptide chain and 21 ■ Amino acids construct the second chains. Both chains are linked by disulfide bond
- 18. ■ Synthesis of human insulin using E.coli Human Insulin is produced by inserting gene of insulin at suitable vector of Escherichia coli (production of Humulin, Eli Lily) by recombinant DNA Technology ■ Selectively obtaining insulin producing beta epithelioid cells from a pancreas, a small circular DNA in a bacteria, called plasmid, modified by inserting human DNA gene for a precursor proinsulin
- 20. ■ Dispersing cells in a nutrient amino acid rich medium composition in an open aeration incubation vessel maintained at about32° -39°C
- 22. Production of recombinant interferon ■ Inside are a group of signalling proteins made and released by host cell in response to the presence of pathogens, such as viruses, bacteria, and tumor cells. ■ The genes of all 3 types Humans have been cloned on micro organisms and expression obtained. ■The problem of both purity and quality resolved by this technology.
- 24. Production of Human growth hormones ■ Somatostatin and somatotrophin are two proteins that act in conjunction to controll growth processes in human body, their malfunctioning leading to painful disorders and also Acromegaly and dwarfism. ■ Same process of insulin, involves insertion of an artificial gene into a lac z vector, synthesis of a fusion protein and cleavage with cyanogen bromide.
- 25. Recombinant blood clotting factors ■ Human factor VIII is a protein that plays a central role in blood clotting. ■ Commenest form of haemophilia in human results from an inability to synthesize factor VIII ■ Production of recombinant human blood clotting factor includes alternative method pharming ■ The complete human C DNA has been attached to the promoter for the acidic protein gene of pig, leading to synthesis of human factor VIII in pig mammary leading to subsequent secretion of protein in milk.
- 26. periplasmic production of recombinant proteins ■ Strategies for the secretory production of recombinant proteins in Escherichia coli. The Sec system, the twin-arginine translocation (TAT) system, and the strategies for enhancing secretory protein production using periplasmic chaperones and protease-negative mutants are shown. A) The co-expression of periplasmic chaperones, such as disulfide -bond formation (Dsb) family proteins, SurA, FkpA, and Skp, can improve the efficiencies of secretory production and protein folding . B) Protease- negative mutant strains can improve secretory production of recombinant proteins by reducing proteolysis . C) A novel TAT system can directly secrete the folded proteins .
- 28. Extracellular production of recombinant proteins ■ Strategies for the extracellular production of recom- binant proteins by E. coli. A) Recombinant proteins can be excreted into the culture medium by treating cells with various agents or by using L-form cells. B) Recombinant protein fused to outer- membrane protein F (OmpF) of E. coli can be excreted into the culture medium . C) Proteins secreted into the E. coli periplasm can also be released into the culture medium by co-expression of kil, out genes, the gene encoding the third topological domain of the transmembrane protein TolA (TolAIII), or the bacteriocin-release protein gene . D) The target protein fused to the C- terminal hemolysin secretion signal can be directly excreted into the culture medium through the
- 30. Application Medicine ■ Therapeutic proteins provide important therapies for a variety of diseases, such as diabetes, cancer, infectious diseases, hemophilia, and anemia. Common therapeutic proteins include antibodies, FC fusion proteins, hormones, interleukins, enzymes, and anticoagulants. There is a growing demand for recombinant proteins for therapeutic applications.
- 31. Therapeutic proteins can be classified into four groups ■ Group I: Therapeutic proteins with enzymatic or regulatory activity. These proteins replace a protein that is deficient or abnormal, up-regulate an existing pathway, or provide a new function or activity. ■ Group II: Therapeutic proteins with special targeting activity. These proteins interfere with a molecule or organism or deliver other molecules. ■ Group III: Therapeutic proteins as vaccines. These proteins help protect against foreign agents, autoimmune diseases, and cancer. ■ Group IV: Therapeutic proteins as diagnostics.
- 32. Research ■ Recombinant proteins help to elucidate the basic and fundamental principles of an organism. These molecules can be used to identify and locate the position of the protein encoded by a specific gene, and to uncover the function of other genes in various cellular activities such as cell signaling, metabolism, growth, replication and death, transcription, translation, and protein modification.
- 33. Biotechnology ■ Recombinant proteins are also used in industry, food production, agriculture, and bioengineering. For example, in breeding industry, enzymes can be added to animal feed to increase the nutritional value of feed ingredients, reduce feed and waste management costs, support animal gut health, enhance animal performance and improve the environment.
- 34. Limitations of recombinant proteins 1. In some cases, the production of recombinant proteins is complex, expensive, and time-consuming. 2. The recombinant proteins produced in cells may not be the same as the natural forms. This difference may reduce the effectiveness of therapeutic recombinant proteins and even cause side effects. Additionally, this difference may affect the results of experiments. 3. A major concern for all recombinant drugs is immunogenicity. All biotechnologically produced therapeutics may exhibit some form of immunogenicity. It is difficult to predict the safety of novel therapeutic proteins.