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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
...
5.2 The Production of Protein

Overview of Protein Synthesis
Protein synthesis occurs continuously
throughout a cell’s lif...
5.2 The Production of Protein

Overview of Protein Synthesis
Protein synthesis occurs continuously
throughout a cell’s lif...
5.2 The Production of Protein

Overview of Protein Synthesis
Protein synthesis occurs continuously
throughout a cell’s lif...
Protein Synthesis in a Eukaryotic Cell.
In a eukaryotic cell, DNA is located within
chromosomes in the nucleus.
Transcript...
Protein Synthesis in a Eukaryotic Cell.
Introns are removed from the RNA and exons
are spliced together.
The exons compris...
Protein Synthesis in a Eukaryotic Cell.
The mRNA transcripts carry the DNA code out
of the nucleus to the ribosomes, which...
Protein Synthesis in a Prokaryotic Cell.
There is no nucleus. There are no introns and
exons and the entire gene codes for...
Transcription and Translation
Protein synthesis is a two-step process:

First Step:
Transcription

Genetic code must
be re...
Transcription and Translation
Protein synthesis is a two-step process:

First Step:
Transcription

mRNA is
complementary t...
Transcription and Translation
Second Step:
Translation
mRNA nucleotide
code is rendered
into a sequence of
amino acids
Translation
CodonA 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
transferase
creates a
peptide bond
linking it to
the growing
polyp...
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.
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.
DNA MUTATIONS- Effect on proteins
Redundancy in the genetic code can
often make DNA point mutations (a
single nucleotide i...
DNA MUTATIONS- Effect on proteins
Frame shift
mutations- inserting
or deleting
nucleotides that are
not multiples of
three...
Post-translational Modifications
Polypeptide chains fold into their 3D
conformations.
The protien may then be modified via
G...
Vocabulary
• Protein synthesis – the generation of new
proteins from amino acid subunits; in the cell, it
includes transcr...
Vocabulary
• tRNA – a type of ribonucleic acid (RNA) that
shuttles amino acids into the ribosome for
protein synthesis
• P...
5.2 Review Questions
1.

2.

3.

Distinguish between transcription and
translation.
If a structural gene’s code is “TAC GG...
Function of Antibody Proteins
Antibodies are proteins that recognize and bind
foreign molecules (antigens) for removal fro...
Function of Antibody Proteins
Invasion by something foreign to the body
(an antigen) stimulates antibody production
by B l...
Function of Antibody Proteins
Antibody proteins recognize a single shape on
an antigen called an epitope and bind there,
h...
Function of Antibody Proteins
Antigens can be
•microorganisms (viruses, bacteria)
•microbial products (toxins)
•foreign pr...
Function of Antibody Proteins
Antibodies are also called immunoglobulins(Ig)
Most is IgG
Function of Antibody Proteins
Epitopes are the specific parts of antigens that
are recognized by antibodies.

•Each antibod...
Function of Antibody Proteins
Epitopes are the specific parts of antigens that
are recognized by antibodies.

•Each antibod...
Function of Antibody Proteins
An HIV virus
particle (virion)
has many
potential epitopes
on its surface that
may be
recogn...
Function of Antibody Proteins

Structure of IgG bound to the HIV capsid protein p24
as determined by X-ray crystallography...
Antibodies are mass produced via many
methods.
polyclonal antibodiesa mixture of antibodies
for a single antigen
monoclona...
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...
ELISA
(Enzyme-Linked Immunosorbent Assay)
-a useful form of analysis that exploits the
amazing specificity of antibodies to...
ELISA
Used for detecting all sorts of molecules and organisms
(Enzyme-Linked Immunosorbent Assay)
-HIV testing or any viru...
Vocabulary

• Antigens – the foreign proteins or
molecules that are the target of binding by
antibodies
• Epitope – the sp...
Vocabulary

• ELISA – short for enzyme-linked
immunosorbent assay, a technique that
measures the amount of protein or anti...
5.1 Review Questions

1.

2.

How many polypeptide chains are
found in an antibody, and how are they
held together in the ...
The Importance of Proteins in
Biotech R&D
The ability to synthesize and modify peptides
or proteins is crucial to the prod...
5.3 Enzymes: Protein Catalysts

Enzymes and Their Substrates
Enzymes are proteins that act as catalysts.
Enzymes are invol...
5.3 Enzymes: Protein Catalysts

Enzymes and Their Substrates
The molecules upon which enzymes act are
called substrates.
5.3 Enzymes: Protein Catalysts

Enzymes and Their Substrates
Enzyme active site and substrate match
exactly (the Lock and ...
5.3 Enzymes: Protein Catalysts

Enzymes and Their Substrates
5.3 Enzymes: Protein Catalysts

Factors That Affect Enzyme Activity

Amount of substrate in the solution
Temperature
Acidi...
5.3 Enzymes: Protein Catalysts

Factors That Affect Enzyme Activity

Amount of substrate in the solution
Temperature
Acidi...
5.3 Enzymes: Protein Catalysts

Factors That Affect Enzyme Activity

All proteins denature in extreme temps
and outside of...
Vocabulary
• Substrate – the molecule that an enzyme acts on
• Lock and key model – a model used to
describe how enzymes f...
Vocabulary

• Optimum temperature – the temperature
at which an enzyme achieves maximum activity
• Denaturation – the proc...
5.4 Studying Proteins

A technician loads protein samples on a vertical gel.
Vertical gel boxes operate in a fashion simil...
Vertical Gel Electrophoresis. Gel cassettes are snapped or
screwed in place (right). Running buffer is added behind the ge...
Silver stain is much more sensitive than Coomassie® Blue. When
samples have low concentrations of protein or DNA, silver-s...
5.5 Applications of Protein Analysis

Protein profile of cells and tissues
A protein’s structure can help explain its
funct...
Questions and
Comments?
Biotechnology Chapter Five Lecture- Proteins (part b)
Biotechnology Chapter Five Lecture- Proteins (part b)
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Biotechnology Chapter Five Lecture- Proteins (part b)

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Biotechnology Chapter Five Lecture- Proteins (part b)
Translation, Enzymes, and Antibodies

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Transcript of "Biotechnology Chapter Five Lecture- Proteins (part b)"

  1. 1. Chapter 5 Lecture (Part Two) Protein production, antibodies, and enzymes
  2. 2. 5.2 The Production of Protein Until recently, proteins could only be made in cells.
  3. 3. 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
  4. 4. 5.2 The Production of Protein Overview of Protein Synthesis Protein 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. 5. 5.2 The Production of Protein Overview of Protein Synthesis Protein 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.
  6. 6. 5.2 The Production of Protein Overview of Protein Synthesis Protein 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.
  7. 7. 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.
  8. 8. 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).
  9. 9. 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.
  10. 10. 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.
  11. 11. Transcription and Translation Protein synthesis is a two-step process: First Step: Transcription Genetic code must be rewritten onto a messenger molecule. RNA polymerase attaches to the promoter region of a gene.
  12. 12. Transcription and Translation Protein synthesis is a two-step process: First Step: Transcription mRNA is complementary to DNA A↬U G↬C T↬A C↬G
  13. 13. Transcription and Translation Second Step: Translation mRNA nucleotide code is rendered into a sequence of amino acids
  14. 14. Translation CodonA group of three nucleotides that codes for one amino acid.
  15. 15. Translation An mRNA usually starts with AUG, the “start” codon. The mRNA attaches to the ribosome here.
  16. 16. Translation tRNA (t for transfer) molecules bring amino acids to the ribosome.
  17. 17. Translation If it is the correctly coded a.a. pedptidyl transferase creates a peptide bond linking it to the growing polypeptide chain.
  18. 18. Translation The ribosome shifts to the next codon. UAA and UAG are the “stop” codons.
  19. 19. Translation The ribosome shifts to the next codon. UAA and UAG are the “stop” codons.
  20. 20. The GENETIC CODE Used for ALL life that we know of!!!
  21. 21. The GENETIC CODE More than one codon for each a.a Often the third nucleotide can vary.
  22. 22. DNA MUTATIONS- Effect on proteins 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 threecause the greatest changes because every a.a. after the mutation will be wrong.
  23. 23. DNA MUTATIONS- Effect on proteins Frame shift mutations- inserting or deleting nucleotides that are not multiples of threecause the greatest changes because every a.a. after the mutation will be wrong.
  24. 24. Post-translational Modifications Polypeptide chains fold into their 3D conformations. The protien may then be modified via Glycosylation- addition of sugar groups Phosphorylation- addition of phosphate groups. Cleavage- cut
  25. 25. Vocabulary • 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
  26. 26. 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
  27. 27. 5.2 Review Questions 1. 2. 3. Distinguish between transcription and translation. If a structural gene’s code is “TAC GGC ATG CCC TTA CGC ATC,” what will the mRNA transcript be? If the mRNA transcript from question No. 2 were translated into a peptide, what would the amino-acid sequence of the peptide be?
  28. 28. Function of Antibody Proteins Antibodies are proteins that recognize and bind foreign molecules (antigens) for removal from the body
  29. 29. Function of Antibody Proteins Invasion by something foreign to the body (an antigen) stimulates antibody production by B lymphocytes (B cell).
  30. 30. Function of Antibody Proteins Antibody proteins recognize a single shape on an antigen called an epitope and bind there, helping immune cells to recognize and attack the antigen.
  31. 31. Function of Antibody Proteins Antigens can be •microorganisms (viruses, bacteria) •microbial products (toxins) •foreign proteins •DNA and RNA molecules •drugs •other chemicals
  32. 32. Function of Antibody Proteins Antibodies are also called immunoglobulins(Ig) Most is IgG
  33. 33. 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.
  34. 34. 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.
  35. 35. Function of Antibody Proteins An HIV virus particle (virion) has many potential epitopes on its surface that may be recognized by many different antibodies.
  36. 36. 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)
  37. 37. Antibodies are mass produced via many methods. polyclonal antibodiesa mixture of antibodies for a single antigen monoclonal antibodiesclones of a single antibody
  38. 38. Antibodies are used for - vaccines - “labeling” molecules for identification
  39. 39. 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.
  40. 40. 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.
  41. 41. ELISA Used for detecting all sorts of molecules and organisms (Enzyme-Linked Immunosorbent Assay) -HIV testing or any virus -a useful form of -drug testing analysis that exploits the amazing specificity of antibodies to their -pregnancy testing -detection of allergens (gluten, soy, peanuts) antigens. -identify bacteria -detect parasites Antibodies are - water contaminants designed toGMO bind -detect specific molecules ELISA can also quantify - Tells HOW MUCH and produce a visible color.
  42. 42. 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
  43. 43. 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)
  44. 44. 5.1 Review Questions 1. 2. How many polypeptide chains are found in an antibody, and how are they held together in the protein? What is the value of monoclonal antibody technology?
  45. 45. 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.
  46. 46. 5.3 Enzymes: Protein Catalysts Enzymes and Their Substrates 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.
  47. 47. 5.3 Enzymes: Protein Catalysts Enzymes and Their Substrates The molecules upon which enzymes act are called substrates.
  48. 48. 5.3 Enzymes: Protein Catalysts Enzymes and Their Substrates Enzyme active site and substrate match exactly (the Lock and Key Model)
  49. 49. 5.3 Enzymes: Protein Catalysts Enzymes and Their Substrates
  50. 50. 5.3 Enzymes: Protein Catalysts Factors That Affect Enzyme Activity Amount of substrate in the solution Temperature Acidity or alkalinity Enzymes have an optimum temperature and pH.
  51. 51. 5.3 Enzymes: Protein Catalysts Factors That Affect Enzyme Activity Amount of substrate in the solution Temperature Acidity or alkalinity Enzymes have an optimum temperature and pH.
  52. 52. 5.3 Enzymes: Protein Catalysts Factors That Affect Enzyme Activity All proteins denature in extreme temps and outside of their optimum pH.
  53. 53. Vocabulary • 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
  54. 54. Vocabulary • Optimum temperature – the temperature at which an enzyme achieves maximum activity • Denaturation – the process in which proteins lose their conformation or threedimensional shape • Optimum pH – the pH at which an enzyme achieves maximum activity
  55. 55. 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.
  56. 56. 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.
  57. 57. 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.
  58. 58. 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
  59. 59. Questions and Comments?
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