This document summarizes the process of designing and purifying proteins, using insulin production as an example. It describes how insulin is produced using recombinant DNA technology by inserting the human insulin gene into E. coli bacteria. The bacteria then act as factories to produce human insulin. The document outlines the various purification techniques used, including cation exchange chromatography and precipitation with zinc, to isolate and purify the insulin protein. It emphasizes the importance of protein design and effective purification protocols for applications in biotechnology and pharmaceutical industries.
METHODS TO DETERMINE PROTEIN STRUCTURE Sabahat Ali
This document discusses several methods for determining protein structure: Edman degradation, X-ray crystallography, Western blotting, SDS-PAGE, 2D gel electrophoresis, and isoelectric focusing. Edman degradation involves chemically removing amino acids from the N-terminus of a protein one by one to determine the sequence. X-ray crystallography provides high-resolution 3D structures of proteins. Western blotting identifies specific proteins in a sample using antibodies. SDS-PAGE and 2D gels separate proteins by size and electric charge properties. Isoelectric focusing separates proteins based on their isoelectric points.
Proteins fold into complex 3D structures essential for their function. There are four levels of protein structure - primary, secondary, tertiary, and quaternary. Chaperone proteins help other proteins fold correctly to prevent aggregation. Misfolded proteins can result from changes in temperature, pH, or lack of chaperones and may lead to disease if not degraded. Normally, misfolded proteins are targeted for degradation by the ubiquitin proteasome pathway, but accumulation of misfolded proteins can cause conditions like Alzheimer's disease.
Tertiary structure describes how protein chains fold upon themselves into complex 3D shapes. These shapes are stabilized by interactions between amino acid side chains like disulfide bonds, hydrogen bonds, and hydrophobic interactions. Long protein chains often contain multiple domains that fold independently. Quaternary structure refers to complexes of two or more protein subunits. Chaperone proteins assist other proteins in proper folding, while misfolded proteins can accumulate and cause diseases.
This document outlines the goals and key concepts regarding protein structure. It discusses the four levels of protein structure - primary, secondary, tertiary, and quaternary. Methods for determining protein structure are also covered, including protein purification techniques like chromatography, electrophoresis, and centrifugation. Protein sequencing methods such as Edman degradation are also summarized. The document provides an overview of protein structure and analysis.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
Site-specific recombination (SSR) systems like Cre/lox and FLP/FRT allow for precise integration of genes of interest (GOI) into plant genomes compared to traditional transformation methods. The Cre/lox system uses the Cre recombinase to catalyze recombination between two loxP sites, while the FLP/FRT system uses FLP recombinase to catalyze recombination between two FRT sites. These systems allow for single-copy, single-locus integration of GOIs without disrupting functional genes. However, constitutive expression of Cre and FLP recombinases can cause unwanted genomic changes. Current research focuses on engineering these recombinases for improved efficiency and versatility of SSR for transgenic plant
1. The document discusses various blotting techniques including Southern blotting, Northern blotting, and Western blotting. It provides a brief history and overview of each technique.
2. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting detects proteins. The techniques involve separating biomolecules by electrophoresis, transferring them to a membrane, then using a probe for detection.
3. The document outlines the basic methodology for each type of blotting. This includes separation, transfer to a membrane, blocking, hybridization with a probe, washing, and detection. Each technique allows for the analysis of biomolecules and has various applications in research.
METHODS TO DETERMINE PROTEIN STRUCTURE Sabahat Ali
This document discusses several methods for determining protein structure: Edman degradation, X-ray crystallography, Western blotting, SDS-PAGE, 2D gel electrophoresis, and isoelectric focusing. Edman degradation involves chemically removing amino acids from the N-terminus of a protein one by one to determine the sequence. X-ray crystallography provides high-resolution 3D structures of proteins. Western blotting identifies specific proteins in a sample using antibodies. SDS-PAGE and 2D gels separate proteins by size and electric charge properties. Isoelectric focusing separates proteins based on their isoelectric points.
Proteins fold into complex 3D structures essential for their function. There are four levels of protein structure - primary, secondary, tertiary, and quaternary. Chaperone proteins help other proteins fold correctly to prevent aggregation. Misfolded proteins can result from changes in temperature, pH, or lack of chaperones and may lead to disease if not degraded. Normally, misfolded proteins are targeted for degradation by the ubiquitin proteasome pathway, but accumulation of misfolded proteins can cause conditions like Alzheimer's disease.
Tertiary structure describes how protein chains fold upon themselves into complex 3D shapes. These shapes are stabilized by interactions between amino acid side chains like disulfide bonds, hydrogen bonds, and hydrophobic interactions. Long protein chains often contain multiple domains that fold independently. Quaternary structure refers to complexes of two or more protein subunits. Chaperone proteins assist other proteins in proper folding, while misfolded proteins can accumulate and cause diseases.
This document outlines the goals and key concepts regarding protein structure. It discusses the four levels of protein structure - primary, secondary, tertiary, and quaternary. Methods for determining protein structure are also covered, including protein purification techniques like chromatography, electrophoresis, and centrifugation. Protein sequencing methods such as Edman degradation are also summarized. The document provides an overview of protein structure and analysis.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
Site-specific recombination (SSR) systems like Cre/lox and FLP/FRT allow for precise integration of genes of interest (GOI) into plant genomes compared to traditional transformation methods. The Cre/lox system uses the Cre recombinase to catalyze recombination between two loxP sites, while the FLP/FRT system uses FLP recombinase to catalyze recombination between two FRT sites. These systems allow for single-copy, single-locus integration of GOIs without disrupting functional genes. However, constitutive expression of Cre and FLP recombinases can cause unwanted genomic changes. Current research focuses on engineering these recombinases for improved efficiency and versatility of SSR for transgenic plant
1. The document discusses various blotting techniques including Southern blotting, Northern blotting, and Western blotting. It provides a brief history and overview of each technique.
2. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting detects proteins. The techniques involve separating biomolecules by electrophoresis, transferring them to a membrane, then using a probe for detection.
3. The document outlines the basic methodology for each type of blotting. This includes separation, transfer to a membrane, blocking, hybridization with a probe, washing, and detection. Each technique allows for the analysis of biomolecules and has various applications in research.
Protein purification techniques involve isolating a single protein from a complex mixture in order to characterize its function, structure, and interactions. The main steps include extracting proteins from cells, stabilizing them in solution, separating them using techniques like precipitation, filtration, dialysis, and various types of chromatography, and then verifying the purity of the isolated protein using analytical methods like SDS-PAGE and mass spectrometry. The basis of protein separation includes differences in solubility, binding interactions, size, charge, and other properties.
Presentation given by Dr. Karthikeyan at Department of Biochemistry, Maulana Azad Medical College.
Addition:
There are certain proteins which are degraded by proteasome without ubiquitin tag. one such example is ornithine decarboxylase - rate limiting enzyme of polyamine synthesis.
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
The document discusses protein synthesis and post-translational modification. It describes how translation involves mRNA, ribosomes, tRNA, and release factors to synthesize proteins. The process involves initiation, elongation, and termination. After synthesis, the peptide undergoes folding, modification like phosphorylation, and can be transported to organelles. Post-translational modifications are important for diversity and regulating protein function, and involve processes like methylation, ubiquitination, and glycosylation. Diseases like atherosclerosis and fibrosis are related to disorders of collagen deposition and modification.
This document discusses the ionization and pH of amino acids. It begins by providing background on amino acids, noting they contain both amine and carboxylic acid functional groups. It then discusses how amino acids can be classified, including by their side chains. Neutral, acidic, and basic side chains are described. PKa values and isoelectric points are also discussed. The document provides examples of glycine ionization and discusses zwitterion formation in amino acids. Tables of amino acid properties including pKa values and molecular weights are also included.
Proteomics and its applications
Proteomics involves the analysis of the entire complement of proteins in a cell, tissue or organism. It assesses protein activities, modifications, localization and interactions. Proteomics uses techniques like gel electrophoresis, mass spectrometry and liquid chromatography to separate and identify proteins. These techniques can be applied to discover disease biomarkers, develop diagnostic tools, and gain insights into disease pathogenesis and treatment. Proteomics has applications in studying various diseases including cancer, diabetes and infections. It provides insights into cellular processes and systems biology.
This document discusses enzymes and enzymology. It begins by defining what an enzyme is and explaining enzyme nomenclature and classification. It then discusses the structural organization of enzymes and different types of cofactors. The mechanisms of enzyme action and factors that affect enzyme kinetics are also explained. Different topics within enzymology are then explored in more detail, including enzyme inhibition mechanisms and how enzyme activity is regulated through various methods like allosteric regulation, covalent modification, and controlling enzyme synthesis and degradation. Clinical applications of enzymes are also briefly covered.
DNA replication is the process by which DNA copies itself for cell division. It is semiconservative, meaning each new DNA molecule contains one old and one new strand. Replication occurs through the unwinding of the DNA double helix at an origin of replication by helicase. DNA polymerase then adds complementary nucleotides to each strand, while ligase seals the DNA. Replication of the leading strand is continuous while the lagging strand occurs discontinuously in fragments called Okazaki fragments. Various enzymes and mechanisms ensure replication occurs with high fidelity and accuracy.
This document discusses homology modeling, which is a computational technique used to develop atomic-resolution models of proteins based on their amino acid sequences and known 3D structures of homologous proteins. It describes the key steps in homology modeling as template identification, target-template alignment, model building and refinement, and model validation. The advantages of homology modeling include that it is faster than experimental techniques. However, the accuracy depends on factors like the sequence identity between the target and template.
Introduction
What is Protein Sequencing?
History
Determination of amino acid composition
Sequencing methods
N terminal sequencing
C terminal sequencing
Mass spectrometer
Application
Reference
This document discusses RNA editing. It defines RNA editing as molecular processes that alter the nucleotide sequence of an RNA molecule after transcription from DNA. There are two main types of RNA editing: substitution editing, where individual nucleotides are chemically altered, and insertion/deletion editing, which adds or removes nucleotides. An example of substitution editing is provided in the human APOB gene, where an enzyme changes a codon resulting in alternative protein isoforms. Guide RNAs are also discussed as facilitating insertion/deletion editing through base-pairing with pre-mRNAs. RNA editing increases proteomic diversity and regulates gene expression.
This document discusses various methods for determining the amino acid sequence of proteins, including:
- Edman degradation, which sequentially removes amino acids from the N-terminus. Up to 60 amino acids can typically be determined.
- Mass spectrometry techniques like MALDI that help determine the mass and sequence of protein fragments.
- Enzymatic cleavage techniques using enzymes like trypsin to break proteins into smaller fragments that can then be sequenced.
Proteolysis, protein degradation and turnoveremicica
This document discusses protein degradation and turnover. It begins by introducing proteolysis, which is the cleavage of peptide bonds by proteases. Limited proteolysis can create new protein products with new functions through posttranslational modification. There are two main types of proteases - exopeptidases that cleave near the ends of proteins, and endopeptidases that cleave internally. Protein degradation involves complete breakdown of proteins into amino acids by multiple cleavages and occurs through two main pathways: lysosomal breakdown in acidic lysosomes using individual proteases, and ubiquitin-dependent breakdown by the proteasome in the cytosol at neutral pH.
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
Protein microarrays allow high-throughput analysis of protein interactions and functions. They consist of large numbers of capture proteins immobilized on a surface to which labeled probe molecules are added to detect reactions by fluorescence. There are analytical arrays to study protein binding properties and functional arrays containing full-length proteins to assay enzymatic activity and detect antibodies. Protein microarrays have applications in diagnostics, proteomics, analyzing protein interactions and functions, antibody characterization, and treatment development.
This document discusses the production of insulin from microbial culture. It begins with an introduction about insulin as a hormone secreted by the pancreas to control blood sugar levels. When insulin is lacking, it causes diabetes mellitus. The main process of industrial insulin production involves genetically engineering microorganisms like E. coli or yeast to produce human insulin through fermentation. Producing insulin this way is significant because it provides a greater supply of higher quality insulin without the risks of animal-sourced insulin, and is more cost-effective for commercial production. The conclusion notes that major companies like Novo Nordisk, Eli Lilly and Sanofi Aventis dominate the insulin market, which has grown at a rate of 7% annually.
This document summarizes key aspects of insulin including its history, structure, biosynthesis, transport, degradation, effects, and clinical correlations. It describes how insulin was first extracted from dog pancreases in 1921. Insulin is a polypeptide hormone composed of two chains that are held together. It regulates carbohydrate, lipid, and protein metabolism. Insulin increases glucose uptake and storage while decreasing gluconeogenesis. Clinically, insufficient insulin production can lead to symptoms of diabetes like frequent urination.
Protein purification techniques involve isolating a single protein from a complex mixture in order to characterize its function, structure, and interactions. The main steps include extracting proteins from cells, stabilizing them in solution, separating them using techniques like precipitation, filtration, dialysis, and various types of chromatography, and then verifying the purity of the isolated protein using analytical methods like SDS-PAGE and mass spectrometry. The basis of protein separation includes differences in solubility, binding interactions, size, charge, and other properties.
Presentation given by Dr. Karthikeyan at Department of Biochemistry, Maulana Azad Medical College.
Addition:
There are certain proteins which are degraded by proteasome without ubiquitin tag. one such example is ornithine decarboxylase - rate limiting enzyme of polyamine synthesis.
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
The document discusses protein synthesis and post-translational modification. It describes how translation involves mRNA, ribosomes, tRNA, and release factors to synthesize proteins. The process involves initiation, elongation, and termination. After synthesis, the peptide undergoes folding, modification like phosphorylation, and can be transported to organelles. Post-translational modifications are important for diversity and regulating protein function, and involve processes like methylation, ubiquitination, and glycosylation. Diseases like atherosclerosis and fibrosis are related to disorders of collagen deposition and modification.
This document discusses the ionization and pH of amino acids. It begins by providing background on amino acids, noting they contain both amine and carboxylic acid functional groups. It then discusses how amino acids can be classified, including by their side chains. Neutral, acidic, and basic side chains are described. PKa values and isoelectric points are also discussed. The document provides examples of glycine ionization and discusses zwitterion formation in amino acids. Tables of amino acid properties including pKa values and molecular weights are also included.
Proteomics and its applications
Proteomics involves the analysis of the entire complement of proteins in a cell, tissue or organism. It assesses protein activities, modifications, localization and interactions. Proteomics uses techniques like gel electrophoresis, mass spectrometry and liquid chromatography to separate and identify proteins. These techniques can be applied to discover disease biomarkers, develop diagnostic tools, and gain insights into disease pathogenesis and treatment. Proteomics has applications in studying various diseases including cancer, diabetes and infections. It provides insights into cellular processes and systems biology.
This document discusses enzymes and enzymology. It begins by defining what an enzyme is and explaining enzyme nomenclature and classification. It then discusses the structural organization of enzymes and different types of cofactors. The mechanisms of enzyme action and factors that affect enzyme kinetics are also explained. Different topics within enzymology are then explored in more detail, including enzyme inhibition mechanisms and how enzyme activity is regulated through various methods like allosteric regulation, covalent modification, and controlling enzyme synthesis and degradation. Clinical applications of enzymes are also briefly covered.
DNA replication is the process by which DNA copies itself for cell division. It is semiconservative, meaning each new DNA molecule contains one old and one new strand. Replication occurs through the unwinding of the DNA double helix at an origin of replication by helicase. DNA polymerase then adds complementary nucleotides to each strand, while ligase seals the DNA. Replication of the leading strand is continuous while the lagging strand occurs discontinuously in fragments called Okazaki fragments. Various enzymes and mechanisms ensure replication occurs with high fidelity and accuracy.
This document discusses homology modeling, which is a computational technique used to develop atomic-resolution models of proteins based on their amino acid sequences and known 3D structures of homologous proteins. It describes the key steps in homology modeling as template identification, target-template alignment, model building and refinement, and model validation. The advantages of homology modeling include that it is faster than experimental techniques. However, the accuracy depends on factors like the sequence identity between the target and template.
Introduction
What is Protein Sequencing?
History
Determination of amino acid composition
Sequencing methods
N terminal sequencing
C terminal sequencing
Mass spectrometer
Application
Reference
This document discusses RNA editing. It defines RNA editing as molecular processes that alter the nucleotide sequence of an RNA molecule after transcription from DNA. There are two main types of RNA editing: substitution editing, where individual nucleotides are chemically altered, and insertion/deletion editing, which adds or removes nucleotides. An example of substitution editing is provided in the human APOB gene, where an enzyme changes a codon resulting in alternative protein isoforms. Guide RNAs are also discussed as facilitating insertion/deletion editing through base-pairing with pre-mRNAs. RNA editing increases proteomic diversity and regulates gene expression.
This document discusses various methods for determining the amino acid sequence of proteins, including:
- Edman degradation, which sequentially removes amino acids from the N-terminus. Up to 60 amino acids can typically be determined.
- Mass spectrometry techniques like MALDI that help determine the mass and sequence of protein fragments.
- Enzymatic cleavage techniques using enzymes like trypsin to break proteins into smaller fragments that can then be sequenced.
Proteolysis, protein degradation and turnoveremicica
This document discusses protein degradation and turnover. It begins by introducing proteolysis, which is the cleavage of peptide bonds by proteases. Limited proteolysis can create new protein products with new functions through posttranslational modification. There are two main types of proteases - exopeptidases that cleave near the ends of proteins, and endopeptidases that cleave internally. Protein degradation involves complete breakdown of proteins into amino acids by multiple cleavages and occurs through two main pathways: lysosomal breakdown in acidic lysosomes using individual proteases, and ubiquitin-dependent breakdown by the proteasome in the cytosol at neutral pH.
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
Protein microarrays allow high-throughput analysis of protein interactions and functions. They consist of large numbers of capture proteins immobilized on a surface to which labeled probe molecules are added to detect reactions by fluorescence. There are analytical arrays to study protein binding properties and functional arrays containing full-length proteins to assay enzymatic activity and detect antibodies. Protein microarrays have applications in diagnostics, proteomics, analyzing protein interactions and functions, antibody characterization, and treatment development.
This document discusses the production of insulin from microbial culture. It begins with an introduction about insulin as a hormone secreted by the pancreas to control blood sugar levels. When insulin is lacking, it causes diabetes mellitus. The main process of industrial insulin production involves genetically engineering microorganisms like E. coli or yeast to produce human insulin through fermentation. Producing insulin this way is significant because it provides a greater supply of higher quality insulin without the risks of animal-sourced insulin, and is more cost-effective for commercial production. The conclusion notes that major companies like Novo Nordisk, Eli Lilly and Sanofi Aventis dominate the insulin market, which has grown at a rate of 7% annually.
This document summarizes key aspects of insulin including its history, structure, biosynthesis, transport, degradation, effects, and clinical correlations. It describes how insulin was first extracted from dog pancreases in 1921. Insulin is a polypeptide hormone composed of two chains that are held together. It regulates carbohydrate, lipid, and protein metabolism. Insulin increases glucose uptake and storage while decreasing gluconeogenesis. Clinically, insufficient insulin production can lead to symptoms of diabetes like frequent urination.
The document compares using LMO (E. coli bacteria) and LMO (Pichia pastoris yeast) to produce insulin and TMOFTM. E. coli is used to produce insulin by transforming it with genes for insulin subunits A and B, while Pichia pastoris is used to express the TMOFTM molecule. Both production methods involve a two-step LMO elimination process using heat and drying to kill the LMOs, verified by culture simulations showing no LMO presence.
Insulin is a hormone produced naturally by the pancreas that regulates blood sugar levels. It is also used as a treatment for diabetes when the body does not produce enough insulin or the insulin is not effective. There are two main methods for producing insulin commercially - through genetically engineered E. coli bacteria by Eli Lilly and through yeast by Nova Nordisk. The processes involve extracting insulin from animal sources or producing it recombinantly and then purifying it through various filtration and crystallization steps to isolate the insulin.
1. The document provides guidance for a tutorial on protein purification techniques for 2nd year biochemistry students.
2. Students are assigned to present on topics related to common purification methods and develop a proposed purification strategy for a specific protein.
3. An overview is given of general considerations for choosing a purification strategy based on a protein's characteristics and useful tools for primary sequence analysis. Chromatography and other methods are discussed at a high level.
Insulin is a hormone that regulates blood glucose levels. It is normally produced by the pancreas but can also be produced synthetically using recombinant DNA technology. There are three main types of synthetic insulin production. Type 1 involves growing the insulin A and B chains separately in E. coli and linking them. Type 2 uses proinsulin processing in E. coli. Type 3 produces analog insulins with modified amino acid sequences. The industrial production process involves an upstream process of cell line preparation in microbes and gene insertion, and a downstream process of fermentation, purification, and packaging. Careful selection of production organism, conditions, equipment and purification methods is needed to successfully manufacture synthetic insulin at scale.
Protein purification involves a series of steps to isolate a single protein from a complex mixture. These steps may separate proteins based on size, charge, or binding affinity. Common techniques include precipitation with ammonium sulfate, chromatography methods like ion exchange, affinity, size exclusion, and reversed-phase chromatography, and electrophoresis. The goal is to free the protein of interest from other materials, separate it from other proteins, and finally isolate it in a pure form for characterization and use.
Industrial production of recombinant human insulinFarzana Sultana
1. The document describes the industrial production process of recombinant human insulin. Researchers insert the human insulin gene into bacteria like E. coli, which then produce human insulin that is purified for use in treating diabetes.
2. There are two main methods - growing the insulin A and B chains separately, or using the proinsulin gene. In both, the gene is inserted into bacteria which are then fermented to produce insulin. Various purification techniques are used to isolate the insulin.
3. Quality control testing ensures the purity and structure of the insulin batches before being approved for use. Future directions include improved drug delivery methods like inhaled insulin, buccal insulin, and insulin patches.
Insulin is a hormone produced by the pancreas that regulates blood sugar levels. It allows glucose in the bloodstream to enter cells and be used for energy. Without insulin, blood sugar builds up and cells are deprived of energy, leading to serious health issues. Diabetes occurs when the body does not produce enough insulin or the cells do not respond properly to insulin. Historically, insulin was purified from animals but is now commonly produced through recombinant DNA technology using modified bacteria. This process involves isolating the human insulin gene, inserting it into bacterial DNA, and causing the bacteria to express and mass produce human insulin.
The document discusses protein purification techniques used in research and industry. It describes a protein expression and purification series that teaches students to express and purify the protein dihydrofolate reductase (DHFR) using different modules, including bacterial cell culture, affinity chromatography using nickel beads, and size exclusion chromatography to remove imidazole. The series mimics real-world protein purification workflows and allows students to purify a non-colored protein and analyze it using techniques like SDS-PAGE electrophoresis and enzymatic assays.
The document describes research into the function of the putative helix-turn-helix protein gp73 in mycobacteriophage HelDan. The researchers created an epitope-tagged version of gp73 and found that it may bind to HelDan genomic DNA based on crosslinking experiments. Future work aims to purify gp73 under native conditions in HelDan-infected M. smegmatis to identify interacting protein partners via mass spectrometry and examine gp73 expression during infection by RT-PCR.
Bioinformatics—an introduction for computer scientistsunyil96
The document introduces computer scientists to the field of bioinformatics. It provides a high-level overview of key concepts, including:
- Bioinformatics aims to develop computational models to complement biological experiments by helping interpret vast amounts of genomic data.
- A living cell can be described at the molecular level, with interactions between intracellular molecules controlled by shape, location, and reactions facilitated by enzymes.
- Computational techniques are needed to understand cell behavior from incomplete and noisy biological data, especially using evolutionary principles to extrapolate information across species.
The document summarizes research into expressing and purifying a recombinant His6-NLS-Cre-MTS protein from E. coli for use in selectively knocking out a gene in the brains of mice. Key points include:
1) The protein contains tags for nickel affinity purification, nuclear localization, and membrane translocation to make it cell-permeable.
2) Issues arose with protein precipitation during dialysis into artificial cerebrospinal fluid (aCSF) that were addressed by trying different aCSF formulations and buffer components.
3) SDS-PAGE analysis showed bands for the desired protein product and possible degradation products from overexpression and purification.
This document provides information on database design and SQL commands. It suggests designing a database for a travel agency with two tables - one to store customer details including a primary key customer ID, and another to store booking details with a foreign key linking it to the customer ID. It then demonstrates how to create tables, insert data, use the SELECT command to view data, and declare primary and foreign keys.
Bioinformatic approaches to functionally characterise RNAsPaul Gardner
This document discusses the growing importance and use of bioinformatics in analyzing large datasets generated by new sequencing and structure determination techniques. It notes the exponential growth in data in genetic databases like Genbank and protein databases like UniProt. It also discusses how bioinformatics is driving many new biological discoveries by enabling analysis of this "data deluge". The document advocates that all biological research can be considered either "physics" by gaining a deep understanding of molecular mechanisms, or "stamp collecting" by just accumulating observations, and that the goal of building a "periodic table" to classify and predict RNA function aims to take an approach grounded in physics-based understanding.
Applying cheminformatics and bioinformatics approaches to neglected tropical ...Sean Ekins
This document summarizes an approach using cheminformatics and bioinformatics to analyze big data related to neglected tropical diseases, specifically applying it to Chagas disease. Key aspects included curating the Trypanosoma cruzi metabolome, developing machine learning models to predict active compounds from screening data, screening over 7,500 compounds and identifying hits, and validating the top 5 hits in vitro and in vivo in a mouse model. One particularly promising hit was pyronaridine, which showed strong anti-trypanosomal activity and is an approved antimalarial, highlighting its potential for repurposing for Chagas disease.
The document describes the database design for a hospital management system. It includes 4 tables - Patient, Doctor, Lab, and Inpatient/Outpatient. It lists the fields, data types, and relationships for each table. It also provides descriptions of common data types like integer, varchar, and date/time. An entity-relationship diagram is included to depict the relationships between entities like patients, doctors, labs, rooms, and bills.
This document discusses database design using the REA data model. It covers the following key points:
1. The steps for designing and implementing a database system using the REA data model include identifying business events, the resources affected by those events, and participating agents.
2. An REA diagram models a business domain by depicting the relationships between resources, events, and agents based on rules like ensuring each event affects a resource and is linked to other events.
3. Developing an REA diagram involves identifying relevant entities from the business, determining the cardinalities of their relationships, and describing how instances of entities relate to each other.
Hemolytic Uremic Syndrome: A Dangerous Complication of E. coliBill Marler
In this presentation provided by the nation's foremost food poison law firm - Marler Clark, Hemolytic Uremic Syndrome (HUS) is explained. HUS is a rare and highly dangerous result of an E. coli infection and can result in acute kidney failure
Golden Rice – A Humanitarian Biotechnology Projectsol777
Presentation of Golden Rice Co-inventor Peter Beyer, professor at the university of Freiburg, at the Comm4Biotech conference 2011 in Strasbourg - more information on http://www.comm4biotech.eu .
Susanne Benner, Max Planck Institute for Chemistry, former BASF Plant Science speaker, gave a keynote speak at the Comm4Biotech conference at the Ecole Supérieure de Biotechnologie de Strasbourg ESBS on the GM food & crop controversy. More information about the conference on http://www.comm4biotech.eu
The Paper: Monsanto coming to Europe is a failed mission. Not only that most GMOs are not allowed in Europe, most of the farmers don't even grow the one that are.
An analysis of misunderstandings.
The presentation: Monsanto coming to Europe is a failed mission. Not only that most GMOs are not allowed in Europe, most of the farmers don't even grow the one that are.
An analysis of misunderstandings.
This document discusses the potential for algae to serve as an energy source. It notes that algae have several advantages over traditional crops for fuel production, including higher photosynthetic efficiency and the ability to grow in saline water. However, challenges remain in developing cost-effective large-scale production methods. Open ponds are currently the most widely used cultivation method but are over 10 times too expensive, while bioreactors can produce high-value products but are over 100 times too expensive for fuel. The document outlines requirements for an algae startup and suggests that further research is still needed to optimize strains and cultivation methods to make algal biofuels commercially viable.
This document discusses the potential for algae to serve as an energy source. It notes that algae have several advantages over traditional crops for fuel production, including higher photosynthetic efficiency and the ability to grow in saline water. However, challenges remain in developing cost-effective large-scale production methods. Open ponds are currently the most widely used cultivation method but are over 10 times too expensive, while bioreactors can produce high-value products but are over 100 times too expensive for fuel. The document outlines requirements for an algae startup and suggests that further research is still needed to optimize strains and cultivation methods to make algal biofuels commercially viable.
2-3 million die every year because of Vitamin A deficiency, 500.000 people get blind, most of them children. With Golden Rice, a lot of these people could be saved. Learn how and why.
2-3 million die every year because of Vitamin A deficiency, 500.000 people get blind, most of them children. With Golden Rice, a lot of these people could be saved. Learn how and why.
Web 2.0 - a buzzword? Learn about rich internet applications, service orientated architectures and the social web in my presentation for my English course.
“An Outlook of the Ongoing and Future Relationship between Blockchain Technologies and Process-aware Information Systems.” Invited talk at the joint workshop on Blockchain for Information Systems (BC4IS) and Blockchain for Trusted Data Sharing (B4TDS), co-located with with the 36th International Conference on Advanced Information Systems Engineering (CAiSE), 3 June 2024, Limassol, Cyprus.
AI-Powered Food Delivery Transforming App Development in Saudi Arabia.pdfTechgropse Pvt.Ltd.
In this blog post, we'll delve into the intersection of AI and app development in Saudi Arabia, focusing on the food delivery sector. We'll explore how AI is revolutionizing the way Saudi consumers order food, how restaurants manage their operations, and how delivery partners navigate the bustling streets of cities like Riyadh, Jeddah, and Dammam. Through real-world case studies, we'll showcase how leading Saudi food delivery apps are leveraging AI to redefine convenience, personalization, and efficiency.
In his public lecture, Christian Timmerer provides insights into the fascinating history of video streaming, starting from its humble beginnings before YouTube to the groundbreaking technologies that now dominate platforms like Netflix and ORF ON. Timmerer also presents provocative contributions of his own that have significantly influenced the industry. He concludes by looking at future challenges and invites the audience to join in a discussion.
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3. INSULIN PRODUCTION
Islet of Langerhans
ORGAN
ORGANISM TISSUE
FUNCTIONS
INFORMATION
DNA CELL (and NUCLEUS)
4. GENETIC INFORMATION OF INSULIN
DNA ≈ Book
CHROMOSOME 11
≈ Chapter
Insulin ≈ Sentence
GENE
CODON
≈ Word
469 letters
C G A T
5. FROM DNA TO INSULIN
Codon
- DNA -
C GA T
- Insulin -
Gl Gl Cy
Gly Ile Val
u n s
Protein = succession of amino acids
Posttranslational
modifications
Insulin correctly folded
functional
7. INSULIN STRUCTURE
469 letters 156 amino acids 51 amino acids.
two chains linked by disulfide bonds
8. INSULIN FUNCTION
Transport of glucose
requires insulin
Type 1 diabetes
Type 2 diabetes
http://www.lillydiabetes.com/content/how-insulin-works.jsp
9. PROTEIN DESIGN
Making entirely new or
modifying proteins for
example as drugs
11. DIFFERENT ADVANTAGES
Bacteria: Yeast: Insect cells Moss cells Mammalian
E.coli S.cerevisae cells
Costs Cheap Cheap More Cheap More
expensive expensive
Setting it up Easy set Relativly More More More
up easy set up complicated complicated complicated
Large scale Easy to Easy to Easy to scale Easy to scale Difficult
production scale up scale up up up
Human-like no To a small Very similar Very similar Very similar
modification extend
in proteins
Multiple No No Yes Yes No
protein
production
13. RECOMBINANT DNA TECHNOLOGY IN
THE SYNTHESIS OF HUMAN INSULIN
Since 1921: Treatement with
insulin derived from animals
Bovine & porcine insulin slightly
different from human insulin
Sometimes inflammation at
injection sites
Fear: long term complications
Solution: Inserting insulin gene
into E.coli to produce identical
human insulin using Recombinant
DNA Technology
15. MANUFACTURING SYNTHETIC HUMAN
INSULIN
Plasmid Plasmid + restriction enzyme
Insertion of the insulin gene
into plasmid (circular DNA)
Restriction enzymes cut
plasmidic DNA
DNA ligase agglutinates the
insulin gene and the plasmidic
DNA
Plasmid + insulin gene
16. MANUFACTURING SYNTHETIC HUMAN
INSULIN
Introduction of recombinant plasmids
into bacteria: E. coli
E.coli = factory for insulin production
Using E. coli mutants to avoid insulin
degradation
Bacterium reproduces the insulin
gene replicates along with plasmid
E. Coli
17. MANUFACTURING SYNTHETIC HUMAN
INSULIN
Formed protein partly of a byproduct the A or B chain of
insulin
Extraction and purification of A and B chains
byproduct byproduct
Insulin A-chain
Insulin B-chain
19. INSULIN PRODUCTION TODAY
Yeast cells as growth medium
Secretion of almost complete human insulin
Minimization of complex and purification
procedures
Yeast Insulin
20. PROTEIN PURIFICATION
Definition
Protein purification is a series of processes intended to isolate a
single type of protein from a complex mixture of proteins
22. DEGREE OF PURITY
Depends on the application of the protein!!!
Industrial
applications: not so strict…
Food and pharmaceuticals
high level required, >99.99%
Degree is set by the FDA (Food and Drug
Administration)
23. PROPERTIES OF PROTEINS USED FOR THE
PURIFICATION
Differences in proprieties allow a separation of different
proteins
Properties come from
Amino acids composition
Amino adic chain length
Structure/shape of the protein
(folding of the amino acid chain)
24. Size
PROPERTIES OF PROTEINS USED FOR Charge
Solubility
THE PURIFICATION
Hydrophobicity
Specific Binding
I. Size proprieties
25. Size
PROPERTIES OF PROTEINS USED FOR Charge
Solubility
THE PURIFICATION
Hydrophobicity
Specific Binding
I. Size proprieties
I. s
II. Charge
+ + - +-- -
- ++ ++- -+-
++ + - + + -+ - - --
+ + - -
+ o -
26. Size
PROPERTIES OF PROTEINS USED FOR Charge
I. S
THE PURIFICATION Solubility
Hydrophobicity
II. . Specific Binding
III. Solubility: pH, T, [Salt] proprieties
-
+ - -
+ +
-
+
-
+ + Salt -
+
-
+
-
+
27. I. S Size
PROPERTIES OF PROTEINS USED FOR Charge
II. . PURIFICATION
THE Solubility
Hydrophobicity
III. . Specific Binding
IV. Hydrophobicity proprieties
28. I. S Size
PROPERTIES OF PROTEINS USED FOR Charge
II. . PURIFICATION
THE Solubility
Hydrophobicity
III. . Specific Binding
IV. Hydrophobicity proprieties
I. S
II. .
III. .
IV. .
V. Specific binding proprieties
29. PROTEIN PURIFICATION
Protein Location Index
intracellular: sonication - Filtration
extracellular - Gel Filtration
Purification: concentrate - Ion Exchange
proteins, seperate chromatography
proteins - Affinity
Filtration and Chromatography
chromatography
31. CHROMATOGRAPHY
Purification using
specifique protein
properties, as: size,
charge, hydrophobicity or
biorecognition
Stationary phase: inert
material, or coated
material
Mobile phase: buffer
32. GEL FILTRATION
Mild conditions
(according to protein)
With any buffer
Isocratic
Porous matrix in the
spherical beads
Small proteins diffuse
into pores, stay longer
33. ION EXCHANGE CHROMATOGRAPHY
IEX
Net surface charge
According to pH and the
number and exposure of
amino acids
Charge = 0 at pI
pH > pI protein –
pH < pI protein +
34. STEPS IN IEX
Matrix with bound
groups that are charged
Equilibration: adjust pH in
order that protein of
interest binds to column
Elution by changing the
ionic strength or the pH
Proteins with highest
charge elute latest
35. AFFINITY CHROMATOGRAPHY
One step
Specific binding between
protein and ligand (eg
substrate, substrate
analogue, inhibitor, cofac
tor)
His tag binds to metal
ions
36. POLY HIS TAG
Commonly used for
recombinant proteins
Ni2+ binds (His)6
Eluting with imidazole
37. INSULIN PURIFICATION
Extraction (separation of Bacteria/Yeasts)
Purification (separation of other proteins) :
Cation exchange chromatography
OD measurement
Precipitation with Zinc
38. INSULIN EXTRACTION
Secretion of insulin in medium: add sequence to insulin gene
Clarification of culture medium: isopropanol added to
medium, centrifugation and filtration
CENTRIFUGATION
Bacteria Medium with
insulin
Medium
get rid of Bacteria/Yeasts
39. INSULIN PURIFICATION
Ex:Cation exchange Chromatography, SP
Sepharose Fast Flow
Resin –CH2SO3-
Total ionic capacity: 180-250μmol/ml gel
Recommended flow rate: 100-300 cm/h
Particle size range: 45-165 μm
Working pH range: 4-13
Maximum temperature: 30°C
40. CATION EXCHANGE CHROMATOGRAPHY
Resin Regeneration: 0.5N NaOH => resin is clean
Equilibration: 20mM sodium citrate buffer at pH 4.0 => fixation
Na+
Mix with insulin diluted with 20mM citrate buffer at pH 4.0 =>
positively charged
Loading of column and flow rate of 200cm/h => fixation of
insulin
X
•CH2 REGENERATION Na+ +
•CH2 ADD MIX •CH2
SO3-
SO3- SO3-
Y EQUILIBRATION
Na+ insulin + +
resin
41. CATION EXCHANGE CHROMATOGRAPHY
Washing: 20mM citrate buffer => elimination of molecules not
fixed
Elution: 100mM tris HCl, pH 7.5 buffer, flow rate of 100cm/h
=> replacement of insulin by H+
+
•CH2 + ELUTION +H
•CH2 •CH2
SO3-
SO3- SO3-
+ Low HCl
concentration + +H
Fraction with
buffer and no
insulin
Fraction with insulin
42. DETERMINATION OF FRACTIONS
CONTAINING INSULINE
OD 280nm
Aromatic amino acid absorb at 280nm => detection of protein
presence in solution
A= εlC ε280nm=0.55 x 104 M-1cm-1
Phenylalanin Tryptophan Tyrosin
43. PRECIPITATION WITH ZINC
Add ZnCl2 to purified insulin and adjust pH to 6 =>
precipitation
Refrigerator (8 °C) for at least 6h
Centrifugation 5000rpm
Drying of pellet => dry insulin
Yield for ion exchange chromatography and
precipitation: around 75%
44. CONCLUSION
Productionof proteins is a big market
Example: Lilly Insulin production since
1923
Nessecity of good design and purification
protocol
Meistens verstecken Forscher die fremden Gene in einem Bakterium. Es dient als \"Vektor\". Auf dem Vektor fährt die Anleitung für den gewünschten Stoff huckepack mit ins Innere der Pflanzenzelle. Dort lösen sich die fremden Gene von ihrem Vehikel und werden in die Pflanzen-DNA eingebaut. Gentechniker zwingen Pflanzen fremdes Erbgut aber auch auf, indem sie DNA an Goldpartikel heften und die Zellen mit dem vergoldeten Erbgut beschießen.
Biomolecules are purified using chromatography techniques that separate them according to differences in their specific properties, as shown in Figure 1. Property Technique Size Gel filtration (GF), also called size exclusion Charge Ion exchange chromatography (IEX) Hydrophobicity Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC) Biorecognition (ligand specificity) Affinity chromatography (AC)
Mild conditions: cold room to 37degree, at any pH, metal ions or co factors, detergents
Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Target protein is collected in a purified, concentrated form.