Biotechnology Overview: rDNA, Cloning, PCR, and Antibodies

Principles: Genomic information is a resource of unparalleled importance for investigators studying any aspect of biology. Genomes vary in size, but all are large enough to direct all aspects of an organism’s structure and function. To approach them often requires tools to break them into small parts that are experimentally digestible.

An organism’s DNA is the ultimate source of biological information

Advances in DNA sequencing are being matched by new approaches to understanding how chromosomal information is expressed and regulated on a genomic and cellular scale. Important clues to protein function are embedded in the sequences of the genes that encode them.

Genomic information is accessible

We can not only elucidate cellular genomic information; we can also change it. That capacity provides a path to altering any aspect of cellular metabolism, structure, or function.

Genomic information is malleable

the complete set of genes or genetic material present in a cell or organism

Genome

the study of the complete set of DNA (including all of its genes) in a person or other organism

• information is a resource of unparalleled importance for investigators studying any aspect of biology

• can vary in size, but all are large enough to direct all aspects of an organism’s structure and function

• To approach them often requires tools to break them into small parts that are experimentally digestible

Genomics

• Considered the basic unit of inheritance

• Genes are passed from parents to offspring and contain the information needed to specify physical and biological traits

Gene

non-translated DNA

introns

coding segments of DNA

exons

selective amplification of a particular gene or DNA segment so that its genetic information may be studied and utilized

DNA cloning

the methods used to accomplish DNA cloning and related tasks

Recombinant DNA technology, genetic engineering

The Process of DNA Cloning: Five general processes

1. Obtain the DNA segment to be cloned

2. Select a small molecule of DNA capable of autonomous replication

3. Join the two DNA fragments covalently

4. Move the recombinant DNA from the test tube to the host organism

5. Select or identify host cells that contain the recombinant DNA

small DNAs capable of autonomous replication (contain origin of replication)

Cloning vectors

composite DNA molecules comprised of covalently linked segments from 2+ sources

Recombinant DNAs

Restriction Endonucleases and DNA Ligases Yield Recombinant DNA

• which enzyme functions to cleave DNA molecules at specific base sequences

Type II Restriction Endonucleases

Restriction Endonucleases and DNA Ligases Yield Recombinant DNA

• which enzyme functions to join two DNA molecules or fragments

DNA Ligase

_____ and ______ restriction endonucleases = large, multisubunit complexes containing both endonuclease and methylase activities

Type I, Type III

require no ATP, and catalyze the hydrolytic cleavage of DNA phosphodiester bonds within the recognition sequence

• They recognize short, usually palindromic, sequences of 4 to 8 base pairs and, in the presence of Mg2+, cleave the DNA within or in close proximity to the recognition sequence

Type II restriction endonucleases

Restriction Endonucleases Leaves _____

• unpaired bases on the ends

• Due to endonucleases making staggered cuts

• Can base pair with each other or complementary sticky ends

sticky ends

Restriction Endonucleases Leaves _____

• no unpaired bases on the ends

• Bases stay paired due to endonucleases making straight cuts

blunt ends

_______ Allow Amplification of Inserted DNA Segments

• Plasmids

• Bacterial artificial chromosomes

• Yeast artificial chromosomes

Cloning Vectors

Circular DNA molecule with 5,000 to 400,000 base pairs

Plasmids

Synthetic plasmid cloning vehicle designed to allow simple and rapid preparation of cloned recombinant DNA fragments

• Commonly used in E. coli

• Key features:

  • Origin of replication (ori) = sequence where replication is initiated

  • Resistance genes (contain information for the production of proteins that make antibiotics ineffective)

  • Recognition sequences for restriction endonucleases

pBR322

laboratory process by which small plasmids are introduced into bacterial cells through heat shock treatment

• Reason why is not well understood

• Becomes less successful as plasmid size increases

Transformation

laboratory process by which small plasmids are introduced into bacterial cells through high-voltage pulses

• Transiently renders the bacterial membrane permeable

Electroporation

A researcher ligates a DNA fragment to pBR322 plasmids, which contain both AmpR and TetR, that were cleaved in the AmpR gene by Pstl. Following ligation and E. coli transformation, colonies with recombinant plasmids will grow on which plates?

Tetracycline plates only

________ Can Be Expressed to Amplify Protein Production

• Purified proteins have many purposes:

  • Elucidating protein function

  • Studying reaction mechanisms

  • Generating antibodies to the proteins

  • Reconstituting complex cellular activities in the test tube with purified components

  • Examining protein binding partners

Cloned Genes

Expressing a Eukaryotic Protein in a Bacterium

• Eukaryotic genes have surrounding sequences needed for their transcription and regulation

• ____= cloning vectors with transcription and translation signals needed for the regulated expression of a cloned gene

Expression vectors

DNA Sequences in a Typical E. coli Expression Vector

• _____ permits regulation by a repressor protein that binds to it

• Promoter allows efficient transcription of the inserted gene

• Transcription-termination sequence sometimes improves the amount and stability of the mRNA produced

• Ribosome-binding site provides sequence signals for the efficient translation of the mRNA derived from the gene

Operator

Many Different Systems Are Used to Express Recombinant Proteins (_______)

• In principle, any organism can serve as a host to express proteins from a different species:

  • Bacteria

  • Yeast

  • Insects and insect viruses

  • Mammalian cells in culture

Expression Systems

• Most common hosts for protein expression

• Prokaryotes, single-celled microorganisms with no nuclei

Bacteria

Advantages of Using ______ Hosts

• Regulatory sequences are well understood

• Can express high levels of cloned proteins

• Easy to store and grow

• Efficient methods for transforming and extracting DNA

• can be grown in huge amounts

Bacterial

Disadvantages of Using Bacterial Hosts

• Some ______ proteins do not fold correctly

• Proteins may not undergo necessary posttranslational modifications or proteolytic cleavage

• Some gene sequences can be difficult to express

• Many eukaryotic proteins aggregate into insoluble cellular precipitates

heterologous

_________ allow for production of proteins.

Expression vectors

_______________: a process that can turn a single copy of a gene into more than a billion copies in just a few hours

• Use of PCR:

  • making lots of DNA for sequencing

• Finding and analyzing DNA from very small samples for use in forensics

• Producing numerous copies of genes for genetic engineering

Polymerase Chain Reaction (PCR)

PCR Process

• PCR results is “exponential” growth (2n copies, where n = # of cycles)

• PCR targets the gene to be copied with primers, single-stranded DNA sequences that are complementary to sequences next to the gene to be copied

• To begin PCR, the DNA sample that contains the gene to be copied is combined with primers that frame the gene on both sides

• DNA polymerase uses the primers to begin DNA replication and copy the gene; the basic steps of PCR are repeated over and over until you have billions of copies of the DNA sequence between the two primers

• ___________ used; is a thermostable DNA polymerase

Taq polymerase

typically involves a series of 20 to 40 repeated temperature changes, called cycles, with each cycle usually comprising three discrete temperature shifts:

1. Denaturation

2. Primer annealing (depending on the primer)

3. Primer extension

Polymerase Chain Reaction (PCR)

• This type of PCR is preceded by a reaction converting RNA into complementary DNA (cDNA); cDNA contains only exons

• The resulting cDNA is used as a template for a second conventional PCR

• The technique is widely used in the detection of RNA viruses and to study gene expression

Reverse Transcriptase PCR (RT-PCR)

PCR-based technique that couples amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction

• Uses fluorescent dye and fluorometer

• Enables calculation of the starting template concentration and is therefore a frequently used analytical tool

Quantitative PCR (qPCR)

glycoproteins belonging to the immunoglobulin (Ig) superfamily that are secreted by B cells to identify and neutralize foreign organisms or antigens

• contain two heavy and two light chains and are grouped into different isotypes dependent on which type of heavy chain they contain

Antibodies (Abs)

• The fragment antigen binding (Fab) portions of the antibody determine its specificity and enable the binding of antigen

• The fragment crystallizable (Fc) portion is responsible for its biological activity; this biological activity depends on interactions between the Fc portion and Fc receptors (FcR) or complement proteins

This region allows antibodies to activate the immune system

IgG Structure

Antigenic determinant

• The portion of an antigen that makes contact with a particular antibody

Epitope

The part of an antibody which recognizes and binds to an antigen

paratope

_______ antibodies refer to a homogenous population of antibodies, which are produced by a single clone of plasma B cells

• They bind to only one epitope on a specific antigen

• low cross reactivity

• Expensive to produce

Monoclonal

________ antibodies refer to a heterogenous mixture of immunoglobulin molecules, which are secreted against a particular antigen; produced by multiple clones of plasma B cells

• Each antibody recognizes different epitopes on the same antigen

• Increased likelihood of cross reactivity

• Less expensive to produce

Polyclonal

Mechanisms of Action of _______

• Antibodies mediate their actions by various types of direct or indirect effects

• 1. Direct modulation of target antigen

  2. Fc binds to Fcγ receptors resulting in antibody-dependent cellular cytotoxicity (ADCC)

  3. Fc binds to complement protein resulting in complement-dependent cytotoxicity (CDC)

  4. Delivery of a cytotoxic agent (drug, radioisotope, toxin)

mAbs

In 1975 _____ and ________ were the first to describe the in vitro production of murine mAbs from hybridomas; hybridomas are immortal antibody-secreting cell lines

Kohler, Milstein

In an attempt to overcome the inherent immunogenicity and reduced effector function of murine mAbs, chimeric mouse-human antibodies (suffix: -_________) were developed

• This was enabled by grafting the entire antigen-specific variable domain of a mouse Ab onto the constant domains of a human Ab using genetic engineering techniques, resulting in molecules that are approximately 65% human

• These chimeric mAbs exhibit an extended half-life and show reduced immunogenicity, but nevertheless, the propensity of chimeric mAbs to induce anti-drug antibodies is still considerable

ximab

To further improve mAb properties, humanized mAbs (suffix: -________) were developed by grafting just the murine hypervariable regions onto a human Ab framework, resulting in molecules that are approximately 95% human

• While humanized mAbs appeared to overcome the inherent immunogenic problems of murine and chimeric mAbs, humanization does have limitations and can be a laborious process

• The advent of in vitro phage display technology and the generation of various transgenic mouse strains expressing human variable domains enabled the generation of fully human mAbs (suffix: -umab)

• Both humanized and fully human mAbs have significantly reduced immunogenic potential and show properties similar to human endogenous IgGs

zumab

Types of mAbs (monoclonal antibodies)

• Murine

omab

Types of mAbs (monoclonal antibodies)

• Chimeric

ximab

Types of mAbs (monoclonal antibodies)

• Humanized

zumab

Types of mAbs (monoclonal antibodies)

• Human

umab

__________ are used for diagnosis, disease treatment and research

• have been used to treat the following conditions:

  • Cancer

  • Organ transplant rejection

  • Inflammatory and autoimmune disorders, including allergies

  • Infections, including COVID-19

  • Osteoporosis

  • Eye conditions

  • Migraines

  • High cholesterol

  • Nervous system disorders

Monoclonal antibodies

§Monoclonal antibodies are given mostly as _________ solution via infusion

intravenous (IV)

One of the most common methods used to produce mAbs

• In this process, antibody-producing B lymphocytes are isolated from mice after immunizing the mice with a specific antigen and are fused with immortal myeloma cell lines to form hybrid cells, called hybridoma cells

• These hybridoma cells are cultured in a lab to produce mAbs

• Antibodies produced are of high purity and are highly sensitive and specific

Hybridoma Technology

Mice that have had DNA from another source put into their DNA; the foreign DNA is put into the nucleus of a fertilized mouse egg and the new DNA becomes part of every cell and tissue of the mouse

Transgenic Mice

Xenomouse

• A transgenic technology

• Antibody-producing B cells, isolated from the spleen of an immunized Xenomouse, are used to produce hybridomas

• Fully human mAbs are produced through the use of hybridoma technology

• ________ (Vectibix), the first therapeutic mAb developed by this transgenic technology was approved by the FDA in 2006 for the treatment of advanced colorectal cancer

Panitumumab

The approval and market introduction of human ______, produced by recombinant technique in Escherichia coli (E. coli), in 1982, represented an important milestone in the field of protein therapeutics

insulin

As therapeutics, they are predominantly used to bind target antigens, which can then block specific signaling pathways or induce cell death; they can also serve as vehicles for the targeted delivery of potent cytotoxic drugs to diseased cells

Antibodies

As therapeutics, they can be delivered to replace a deficient or absent enzyme in enzyme replacement therapy; in other cases, they can be used as agents to catalyze the degradation, cleavage, or chemical modification of therapeutically relevant targets

Enzymes

As therapeutics, these proteins can be plasma-derived or produced recombinantly and administered to patients with a deficiency in native levels

Coagulation factors

Common Classes of Protein-based Therapeutics

antibodies, enzymes, coagulation factors, protein hormones, cytokines

Act as chemical messengers by binding to receptors which then trigger a signaling cascade and lead to a physiological response; insulin represents a classic example

Protein hormones

Encompasses molecules such as interleukins, interferons, and colony-stimulating factors that mediate cell-to-cell communication during immune responses; this contrasts with protein hormones, which largely regulate the endocrine system

• can be used as immunomodulatory agents for a variety of indications, including multiple sclerosis and leukemia

Cytokines

Protein Stability

• Understanding stability implies knowing the mechanisms that lead to instability of the protein drug

• Leads to determining factors influencing the “shelf life” or stability of the protein drug

• Thus, the study of protein stability in essence is the study of factors/pathways that lead to protein instability

• Protein instability can be classified into two major categories:

Chemical instability, Physical instability

Making or breaking covalent bonds resulting in a new chemical entity

• Types of chemical modification:

  • Hydrolysis

    • Side chain hydrolysis - Deamidation

    • Peptide bond hydrolysis - Proteolysis (fragmentation)

  • Oxidation

  • Racemization

  • Disulfide exchange reactions

chemical instability

Involves structural changes that are independent of chemical modifications

• Types of physical instability:

  • Denaturation

  • Aggregation

  • Precipitation

  • Surface Adsorption

Physical Instability

• Most proteins in native state are globular

• Proteins fold (adopt globular structure) to reduce the exposure of hydrophobic groups to the surrounding environment

  • i.e., Hydrophobic amino acids are generally inside while hydrophilic amino acids are on the surface of the protein

• The loss of globular structure (unfolding) of a protein is termed _______

• Caused by various factors including heat, pH changes, etc.

• Denatured proteins generally result in loss of biological activity

denaturation

Self-association of protein molecules resulting in the formation of dimers, trimers, tetramers, etc. and macromolecular intermediates

• Partially unfolded proteins (through denaturation) can lead to ______

• increases the likelihood of adverse immunogenic effects

• Increases patient morbidity and mortality

• Loss of biological function of the protein; thus, reducing efficacy

• Imparts turbidity to solution or physical separation

• may be reversible and _ may be soluble

aggregation

The extreme form of aggregation (macroscopic aggregation)

• It is irreversible, and the protein is partially or completely unfolded

• Not all insoluble protein material is due to aggregation (e.g., salting-out effect)

• Salted-out proteins still retain activity and native-like structure and the _____ is fully reversible upon dilution

precipitation

Adhesion of proteins to surfaces

• Proteins can adhere to the walls of the vial by surface adsorption and can “flocculate or frost” leading to problems in bio-equivalence (dose delivered)

• Surface-induced protein instability begins with:

  • Adsorption of either native or partially unfolded protein on the surface

  • Surface tension forces at various interfaces can drive aggregation by affecting structural integrity of protein molecules

  • Desorption of partially unfolded proteins from the surface can lead to nucleation and growth of aggregates in the bulk solution

Surface Adsorption

One Way to Improve Protein Stability – ________ (Freeze-drying

• Removal of moisture (solvent) from a frozen product under vacuum

• Essentially, it is low temperature dehydration process, which involves freezing the product, lowering the pressure, then removing the ice by sublimation

• Occurs in three stages:

  • Freezing

  • Primary drying

  • Secondary drying

• Reactions that can occur to the product in the liquid phase such as hydrolysis, cross-linking, oxidation, aggregation, and disulfide rearrangement are essentially eliminated when the product is frozen dried

• Freeze drying is also advantageous because the dry product has a very high specific surface area, which leads to a rapid and complete redissolution

Lyophilization

Lyophilization, Step 1. Freezing

• Generation of ice-water interface

• Proteins can denature at the ice-water interface

• ________ - Excipients used to protect the protein from stresses during freezing

  • E.g., Disaccharides (sucrose), glycerol, and polyethylene glycol (PEG)

Cryoprotectants

Lyophilization, Step 2. Primary Drying

• The ice formed during freezing is removed by ______ at sub-ambient temperatures under vacuum

• Typically carried out at chamber pressures of 40-400 Torr and shelf temperatures ranging from –30 to 10 °C

sublimation

Lyophilization, Step 3. Secondary Drying

• Relatively small amount of bound water remaining in the matrix is removed by _______

• During this stage, the temperature of the shelf and product are increased to promote adequate desorption rates and achieve the desired residual moisture

  • Removal of water during drying stages can cause destabilization of the protein structure

  • Lyoprotectants (e.g., sucrose, trehalose) can act as water substitute and form H-bonding with the proteins and stabilize during drying

  • Lyoprotectants also form glasses of extremely high viscosity; the drug and water molecules are immobilized in the viscous glass, leading to extremely high activation energies required for any reactions to occur

desorption