Biotechnology Chapter Five Lecture- Proteins (part b)

Chapter 5 Lecture (Part Two)

Protein production, antibodies, and enzymes

5.2 The Production of Protein

Until recently, proteins could only be made in cells.

5.2 The Production of Protein

Until recently, proteins could only be made in cells.

Now small polypeptide

chains can be synthesized in the laboratory.

Argonaut Technology Quest 210 Protein

Synthesizer

5.2 The Production of Protein

Overview of Protein SynthesisProtein synthesis occurs continuously

throughout a cell’s life.

Protein synthesis is similar for eukaryotes and prokaryotes.

Protein synthesis occurs on ribosomes through a process

called translation.

5.2 The Production of Protein

Overview of Protein SynthesisProtein synthesis occurs continuously

throughout a cell’s life.

Protein synthesis is similar for eukaryotes and prokaryotes.

Protein synthesis occurs on ribosomes through a process

called translation.

5.2 The Production of Protein

Overview of Protein SynthesisProtein synthesis occurs continuously

throughout a cell’s life.

Protein synthesis is similar for eukaryotes and prokaryotes.

Protein synthesis occurs on ribosomes through a process

called translation.

Protein Synthesis in a Eukaryotic Cell. In a eukaryotic cell, DNA is located within

chromosomes in the nucleus. Transcription- RNA is transcribed from the

DNA template.

Protein Synthesis in a Eukaryotic Cell. Introns are removed from the RNA and exons

are spliced together.The exons comprise the mRNA (m for

messenger).

Protein Synthesis in a Eukaryotic Cell. The mRNA transcripts carry the DNA code out of the nucleus to the ribosomes, which translate

the code into a strand of amino acids.

Protein Synthesis in a Prokaryotic Cell. There is no nucleus. There are no introns and exons and the entire gene codes for protein.

Translation can begin before the mRNA is even done being transcribed.

Genetic code must be rewritten onto a

messenger molecule.

RNA polymerase attaches to the

promoter region of a gene.

First Step: Transcription

Protein synthesis is a two-step process:Transcription and Translation

mRNA is complementary to

DNA

A ↬ U

G ↬ C

T ↬ A

C ↬ G

First Step: Transcription

Protein synthesis is a two-step process:Transcription and Translation

mRNA nucleotide code is rendered

into a sequence of amino acids

Second Step: Translation

Transcription and Translation

Codon- A group of

three nucleotides

that codes for one amino

acid.

Translation

An mRNA usually starts with AUG, the “start” codon.

The mRNA attaches to the ribosome here.

Translation

tRNA (t for transfer)

molecules bring amino acids to the ribosome.

Translation

If it is the correctly coded a.a.pedptidyl

transferasecreates a

peptide bond linking it to the growing polypeptide

chain.

Translation

The ribosome shifts to the next codon.

UAA and UAG are the “stop”

codons.

Translation

The ribosome shifts to the next codon.

UAA and UAG are the “stop”

codons.

Translation

The GENETIC CODE

Used for ALL life that we

know of!!!

The GENETIC CODE

More than one codon

for each a.a

Often the third

nucleotide can vary.

Redundancy in the genetic code can often make DNA point mutations (a

single nucleotide is changed) irrelevant.

Frame shift mutations- inserting or deleting nucleotides that are not

multiples of three-cause the greatest changes because every a.a. after the mutation will be

wrong.

DNA MUTATIONS- Effect on proteins

DNA MUTATIONS- Effect on proteins

Frame shift mutations- inserting

or deleting nucleotides that are

not multiples of three-

cause the greatest changes because

every a.a. after the mutation will be

wrong.

Post-translational Modifications

Polypeptide chains fold into their 3D conformations.

The protien may then be modified viaGlycosylation- addition of sugar groupsPhosphorylation- addition of phosphate groups.Cleavage- cut

•Protein synthesis – the generation of new proteins from amino acid subunits; in the cell, it includes transcription and translation

•Transcription – the process of deciphering a DNA nucleotide code and converting it into RNA nucleotide code; the RNA carries the genetic message to a ribosome for translation into a protein code

•Codon – a set of three nucleotides on a strand of mRNA that codes for a particular amino acid

•Translation – the process of reading a mRNA nucleotide code and converting it into a sequence of amino acids

Vocabulary

• tRNA – a type of ribonucleic acid (RNA) that shuttles amino acids into the ribosome for protein synthesis

•Peptidyl transferase – an enzyme found in the ribosome that builds polypeptide chains by connecting amino acids into long chains through peptide bonds

•Phosphorylation – adding phosphate groups

•Cleavage – process of splitting the polypeptide into two or more strands

Vocabulary

1. Distinguish between transcription and translation.

2. If a structural gene’s code is “TAC GGC ATG CCC TTA CGC ATC,” what will the mRNA transcript be?

3. If the mRNA transcript from question No. 2 were translated into a peptide, what would the amino-acid sequence of the peptide be?

5.2 Review Questions

Antibodies are proteins that recognize and bind foreign molecules (antigens) for removal from

the body

Function of Antibody Proteins

Function of Antibody ProteinsInvasion by something foreign to the body (an antigen) stimulates antibody production by B lymphocytes (B cell).

Function of Antibody ProteinsAntibody proteins recognize a single shape on an antigen called an epitope and bind there, helping immune cells to recognize and attack the antigen.

Antigens can be •microorganisms (viruses, bacteria)•microbial products (toxins)•foreign proteins•DNA and RNA molecules•drugs•other chemicals

Function of Antibody Proteins

Antibodies are also called immunoglobulins(Ig)

Function of Antibody Proteins

⬅Most is IgG

Epitopes are the specific parts of antigens that are recognized by antibodies. •Each antibody recognizes a single epitope.

Multiple antibodies may recognize and bind to different epitopes on a single antigen.

Function of Antibody Proteins

Epitopes are the specific parts of antigens that are recognized by antibodies. •Each antibody recognizes a single epitope.

•Multiple antibodies may recognize and bind to different epitopes on a single antigen.

Function of Antibody Proteins

An HIV virus particle (virion) has many potential epitopes on its surface that may be recognized by many different antibodies.

Function of Antibody Proteins

Function of Antibody Proteins

Structure of IgG bound to the HIV capsid protein p24 as determined by X-ray crystallography. (Harris et al.1998, Momany et al. 1996)

Antibodies are mass produced via many methods.

polyclonal antibodies- a mixture of antibodies for a single antigen

monoclonal antibodies- clones of a single antibody

Antibodies are used for- vaccines- “labeling” molecules for identification

ELISA (Enzyme-Linked Immunosorbent Assay)-a useful form of analysis that exploits the amazing specificity of antibodies to their antigens.

Antibodies are designed to bind specific molecules and produce a visible color.

ELISA (Enzyme-Linked Immunosorbent Assay)

-a useful form of analysis that exploits the amazing specificity of antibodies to their antigens.

Antibodies are designed to bind specific molecules and produce a visible color.

ELISA (Enzyme-Linked Immunosorbent Assay)

-a useful form of analysis that exploits the amazing specificity of antibodies to their antigens.

Antibodies are designed to bind specific molecules and produce a visible color.

Used for detecting all sorts of molecules and organisms-HIV testing or any virus

-drug testing-pregnancy testing

-detection of allergens (gluten, soy, peanuts)-identify bacteria-detect parasites

- water contaminants-detect GMO

ELISA can also quantify - Tells HOW MUCH

Vocabulary

•Antigens – the foreign proteins or molecules that are the target of binding by antibodies

•Epitope – the specific region on a molecule that an antibody binds to

Vocabulary

•ELISA – short for enzyme-linked immunosorbent assay, a technique that measures the amount of protein or antibody in a solution

•Monoclonal antibody – a type of antibody that is directed against a single epitope

•Hybridoma – a hybrid cell used to generate monoclonal antibodies that results from the fusion of immortal tumor cells with specific antibody-producing white blood cells (B-cells)

5.1 Review Questions

1. How many polypeptide chains are found in an antibody, and how are they

held together in the protein?

2. What is the value of monoclonal antibody technology?

The Importance of Proteins in Biotech R&D

The ability to synthesize and modify peptides or proteins is crucial to the production of

virtually every biotechnology product.

5.3 Enzymes: Protein Catalysts

Enzymes are proteins that act as catalysts.

Enzymes are involved in virtually every reaction in a cell.

Many companies have focused on producing enzymes for sale.

Enzymes and Their Substrates

5.3 Enzymes: Protein Catalysts

The molecules upon which enzymes act are called substrates.

Enzymes and Their Substrates

5.3 Enzymes: Protein Catalysts

Enzymes and Their Substrates

Enzyme active site and substrate match exactly (the Lock and Key Model)

5.3 Enzymes: Protein Catalysts

Enzymes and Their Substrates

5.3 Enzymes: Protein Catalysts

Factors That Affect Enzyme Activity

Amount of substrate in the solutionTemperature

Acidity or alkalinity

Enzymes have an optimum temperature and pH.

5.3 Enzymes: Protein Catalysts

Factors That Affect Enzyme Activity

Amount of substrate in the solutionTemperature

Acidity or alkalinity

Enzymes have an optimum temperature and pH.

5.3 Enzymes: Protein Catalysts

Factors That Affect Enzyme Activity

All proteins denature in extreme temps and outside of their optimum pH.

•Substrate – the molecule that an enzyme acts on

• Lock and key model – a model used to describe how enzymes function, in which the enzyme and substrate make an exact molecular fit at the active site, triggering catalysis

• Induced fit model – a model used to describe how enzymes function, in which a substrate squeezes into an active site and induces the enzyme’s activity

Vocabulary

•Optimum temperature – the temperature at which an enzyme achieves maximum activity

•Denaturation – the process in which proteins lose their conformation or three-dimensional shape

•Optimum pH – the pH at which an enzyme achieves maximum activity

Vocabulary

5.4 Studying Proteins

A technician loads protein samples on a vertical gel. Vertical gel boxes operate in a fashion similar to

horizontal gel boxes.

Vertical Gel Electrophoresis. Gel cassettes are snapped or screwed in place (right). Running buffer is added behind the gel,

covering the wells. Buffer is poured in the front of the gel cassette to cover the front opening. When the top is placed on the box (left) and the power is turned on, electricity flows from the top

(negative charge) to bottom (positive charge). Negatively charged samples move down the gel toward the positive electrode.

Silver stain is much more sensitive than Coomassie® Blue. When samples have low concentrations of protein or DNA, silver-staining

is the method of choice.

5.5 Applications of Protein Analysis

Protein profile of cells and tissues

A protein’s structure can help explain its function

Study chemical processes in cells

Evolutionary and taxonomic relationships

Questions and Comments?