Proteins as Products

1
Chapter 4

• Proteins as Products

2
Learning Objectives
3
(No Transcript)
4
Introduction

• Proteins are large molecules that are required
  for the structure, function, and regulation of
  living cells
• Unique functions to sustain life
• In 2000, the National Institutes of Health
  launched the Protein Structure Initiative to
  identify the structure of human proteins.
• 1,200 protein structures have been identified
  thus far
• The public database hold more than 33,000 protein
  sequences and only 5 have been structured

5
(No Transcript)
6
Proteins as Biotechnology Products

• Many food-processing applications depend on
  proteins
• Beer brewing and winemakingenzymes from yeast
• Cheese-making
• Initially the industry used calves stomachs for
  proteins
• Now use genetically engineered bacteria
• Main enzymes for food production are ones that
  break down large molecules called
  depolymerization
• Amylase
• Proteases
• Lipases
• Other protein include hormones and antibodies for
  medical industry

7
(No Transcript)
8
4.2 Proteins as Biotechnology Products

• Making a Biotech Drug
• Monoclonal antibodies, blood proteins, enzymes
  to fight disease
• Normally not synthetically produced
• Produced through microbial fermentation or
  mammalian cell culture.
• 40 are currently in use.
• 400 new biotechnology medicines in production.
  Most are proteins

9
Producing a Biotech Drug

• Complicated and time-consuming process
• Produce large batches by growing host cell that
  have been transformed in a bioreactor
• Must strictly comply with FDA regulations at all
  stages of the procedure

10
4.2 Proteins as Biotechnology Products

• Applications of Proteins in Industry
• Medical applications
• Food processing
• Textiles and leather goods
• Detergents
• Paper manufacturing and recycling
• Adhesives natural glues
• Bioremediation treating pollution with proteins

11
Medical Applications

• Biotechnology proteins have revolutionized the
  health care and pharmaceutical industries in the
  past several decades
• Diabetes and Gauchers disease can be treated by
  replacing missing proteins
• Diabetes
• Insulin was harvested from pigs or cows. Human
  bodies normally rejected the foreign protein
• Now use E. Coli. Remember the first recombinant
  DNA drug

12
(No Transcript)
13
Example of medical applications

• Treatment of Gauchers disease
• Rare disorder
• Mutation results in the buildup of fats in the
  organs, including the brain
• Fatal if untreated
• Current treatment is costly
• Human placentas are the only source of the enzyme
• 400-2000 placenta are required for a single dose
• Now with biotechnology. Use genetically altered
  tobacco plants to produce the enzyme.

14
(No Transcript)
15
(No Transcript)
16
Food Processing

• Proteins are used to improve
• Baby food
• Canned fruit
• Cheeses
• Desserts
• Dietetic foods equal
• Bread- proteins used to make bread rise quicker
• Soft drinks have longer shelf life
• etc

17
Textiles and Leather Goods

• Enzymes are now replacing harsh chemicals used to
  lighten and soften fabrics
• Enzymatic bio-bleaches reduced demand for
  ordinary bleaches
• Clean wool and soften it.
• Leather is normally processed with enzymes
• Remove hair and

18
Detergents

• Enzymes help cleaning and are biodegradable
• Laundry detergents use enzymes such as proteases,
  lipases, and amylases to dissolve stains in
  cooler water.
• Enzymes are normally the active ingredient

19
Paper Manufacturing and Recycling

• Harmful bleaches were used in the past to lighten
  paper
• Now enzymes are used to do the job

20
Adhesives Natural Glues

• Natural protein adhesives are strong and water
  insoluble
• Nontoxic, biodegradable, and rarely trigger an
  immune response
• Used in medical field to reattach tendons and
  tissues, fill cavities in teeth, and repair
  broken bones

21
Bioremediation Treating Pollution

• Proteins are used to clean up harmful wastes
• Use genetically engineered bacteria to clean up
  waste (oil)
• Neutralizing heavy metal pollutants like
  mercury and cadmium. Harm food chain and normally
  resist enzymatic breakdown
• Now genetically engineer microorganisms that have
  a sticky coat of metallothioneins, proteins that
  capture heavy metals
• Pollutant is not digested but simply made less
  dangerous

22
4.3 Protein Structures

• Proteins
• Are complex molecules built of chains of amino
  acids
• Have electrical charge that causes them to
  interact with other atoms and molecules
• Hydrophilic water loving
• Hydrophobic water hating

23
4.3 Protein Structures

• Structural Arrangement four levels
• Primary structure is the sequence in which amino
  acids are linked together
• Secondary structure occurs when chains of amino
  acids fold or twist at specific points
• Alpha helices and beta sheets
• Tertiary structures are formed when secondary
  structures combine and are bound together
• Quaternary structures are unique, globular,
  three-dimensional complexes built of several
  polypeptides

24
4.3 Protein Structures
25
4.3 Protein Structures

• Protein Folding
• The structure and function of a protein depends
  on protein folding
• If protein is folded incorrectly, desired
  function of a protein is lost and a misfolded
  protein can be detrimental
• 1951 two regular structures were described
• Alpha helices and beta sheets
• Structures are fragile hydrogen bonds are easily
  broken

26
4.3 Protein Structures

• Glycosylation post-translational modification
  wherein carbohydrate units are added to specific
  locations on proteins
• More than 100 post-translational modifications
  occur
• Most common is glycosylation sugar is added
• Change can significantly affect protein activity
• Other modification phosphorylation, sulfur,
  acetylation, etc.

27
4.3 Protein Structures

• Protein Engineering
• Introducing specific, predefined alterations in
  the amino acid sequence through a process known
  as directed molecular evolution technology
• Creating entirely new protein molecules
• Induces mutations randomly into genes and then
  selects the organisms (bacteria) with the protein
  product (enzyme) that has the highest activity.
• Some cases tolerate 1.0M cyanide concentration
• This would have never occurred during natural
  molecular evolution
• These organisms can be used to remediate cyanide
  contamination

28
(No Transcript)
29
New proteins

• Create entirely new protein molecules
• Invent proteins to study certain diseases.
  Develop detection and control methods
• Example
• Prions infectious protein particles caused by
  faulty protein folding
• Attract normal cell proteins and induce changes
  in their structure
• Damaged protein accumulates
• Scrapie in sheeps and goats, mad cow in cows
• Humans kuru and transformable spongiform
  encephalitis (TSE)

30
(No Transcript)
31
4.4 Protein Production

• Proteins are valuable
• Proteins are complex and fragile products
• Production of proteins is a long and painstaking
  process
• Two Main processes
• Upstream processing includes the actual
  expression of the protein in the cell
• Downstream processing the protein is first
  separated from other parts of the cells and
  isolated from other proteins. This involves
  purification of the protein and verification of
  the function a stable means of preserving the
  protein is also required

32
4.4 Protein Production

• Protein Expression The First Phase in Protein
  Processing
• Selecting the cell to be used as a protein source
• Microorganisms
• Fungi
• Plants
• Mammalian cell systems
• Whole-animal production systems
• Insect systems

33
(No Transcript)
34
(No Transcript)
35
(No Transcript)
36
4.5 Protein Purification Methods

• Protein Must Be Harvested
• Entire cell is harvested if protein is
  intracellular
• Requires cell lysis to release the protein
• Releases the entire contents of the cell
• Culture medium is collected if the protein is
  extracellular

37
4.5 Protein Purification Methods

• Downstream processing after the protein is
  produced
• If protein is intracellular the entire cell is
  harvested
• If extracellular, the medium is collected
• FDA required 99.9 pure protein
• Separating the Components in the Extract
• Similarities between proteins allow the
  separation of proteins from non-protein material
• Protein precipitation salts cause proteins to
  settle out of solution
• Filtration (size-based) separation methods
• Centrifugation
• Membrane filtration
• Microfiltration
• Ultrafiltration

38
Preparing the Extract for Purification

• Collect medium or lyze the cell.
• Cell lysis can be done by freezing and thawing
  the cells, detergents, mechanical ultrasonics or
  grinding
• After disruption add detergents or salts
• Detergents reduce the hydrophobic orientation of
  the proteins needed to separate by size (SDS)
• Salts reduce interactions between the molecules
  and keep the proteins in solution
• Proteins must be stabilized.
• Remember temperature denatures proteins
• Proteases digest proteins
• Foaming or shearing proteins

39
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Goal is to separate proteins from nonprotein
  material such as lipids, carbohydrates, and
  nucleic acids
• Protein precipitation
• Salts are added to precipitate protein
• Interact with hydrophilic amino acids

40
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Differences in proteins allows the separation of
  the target protein from other proteins
• Chromatography allows the sorting of proteins
  based on size or by how they cling to or dissolve
  in various substances

41
Filtration (size-based separation)

• Centrifugation
• Use high speed
• Proteins are isolated in a single layer
• Membrane filtration
• Thin membranes of nylon with small pores used to
  filter out cellular debris and leave protein
• Dialysis

42
(No Transcript)
43
(No Transcript)
44
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Chromatography
• Sort out the proteins by size or how they cling
  to various substances
• Size exclusion chromatography (SEC) uses gel
  beads with pores
• Larger proteins move quickly around the beads and
  smaller proteins slip through the pores and
  therefore move more slowly through the beads

45
4.5 Protein Purification Methods
46
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Chromatography
• Ion exchange chromatography relies on the
  charge of the protein
• Resin is charged
• Opposite charged proteins will stick to resin
  beads
• Can be eluted by changing the charge with salts
  of increasing concentration

47
4.5 Protein Purification Methods
48
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Chromatography
• Affinity chromatography relies on the ability of
  proteins to bind specifically and reversibly to
  uniquely shaped compounds called ligands

49
(No Transcript)
50
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Chromatography
• Hydrophobic interaction chromatography (HIC)
  sorts proteins on the basis of their repulsion of
  water

51
4.5 Protein Purification Methods
52
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Iso-electric focusing used in QC to identify two
  similar proteins that are difficult to separate
  by any other means
• Each protein has a specific number of charged
  amino acids on its surface in specific places
• Creates a unique electric signature known as its
  iso-electric point (IEP) where charges on the
  protein match the pH of the solution

53
4.5 Protein Purification Methods

• Separating the Components in the Extract
• Analytic methods
• High-Performance liquid chromatography (HPLC)
  uses high pressure to force the extract through
  the column in a shorter time
• Mass spectrometry (mass spec) highly sensitive
  method used to detect trace elements
• Used to indicate the size and identity of most
  protein fragments

54
4.6 Verification

• The presence and concentration of the protein of
  interest must be verified at each step of the
  purification process
• SDS-PAGE (polyacrylamide gel electrophoresis)
• Western blotting
• ELISA

55
4.7 Preserving Proteins

• Lyophilization (freeze-drying)
• Protein, usually a liquid product, is first
  frozen
• A vacuum is used to hasten the evaporation of
  water from the fluid
• Will maintain protein structure and can be stored
  at room temperature for long periods of time

56
4.8 Scale-Up of Protein Purification

• Protocols are usually designed in the laboratory
  on a small scale
• Must be scaled up for production
• Process is approved by FDA so must make sure
  laboratory procedures can be scaled up

57
4.9 Postpurification Analysis Methods

• Protein Sequencing
• Must determine the primary structure, the
  sequence of amino acids
• X-ray Crystallography
• Used to determine the complex tertiary and
  quaternary structures

58
4.10 Proteomics

• A new scientific discipline dedicated to
  understanding the complex relationship of disease
  and protein expression
• Uses protein microarrays to test variation in
  protein expression between healthy and disease
  states