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Biotech (Recomb. Protein Production) > Exam 1 > Flashcards

Exam 1 Flashcards

1
Q

Steps of Recombinant DNA

A

1) Gene of Interest
2) Expression Vector
3) Enzymes
4) Transformation (or Transfection)
5)Screening & Selection
6) rDNA Cloning
7) Protein Production

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2
Q

Expression Vectors

A

1) Plasmid (smallest)
2) Bacteriophage lambda
3) Bacteriophage P1
4) BAC (Bacteria Artificial Chromosome)
5) P1 Bacteriophage-Derived Artificial Chromosome
6) Yeast Artificial Chromosome
7) Human Artificial Chromosome (largest)

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3
Q

Requirements of Plasmids

A

1) Replication Origin
2) Selection Marker (ex. AMP-r)
3) MCS (multiple cloning sites) or RE (restriction enzymes)

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4
Q

Genomic Library

A

A set of thousands of DNA segments from a genome, each carried by a plasmid, phage, or other cloning vector.

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5
Q

Lysogony

A

A type of life cycle that takes place when a bacteriophage infects certain types of bacteria (bacteriophage chromosome integrates with host cell’s chromosome)

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6
Q

Lytic cycle

A

Bacteriophage replication cycle resulting in the release of new phages by lysis (and death) of the host cell.

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7
Q

Commonly Used Enzymes

A

Restriction enzymes that are able to recognize a certain sequence and leave either a 5’ basic overhang (ex. EcoRI) or blunt ends (ex. HindIII)

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8
Q

Neoschizomers

A

A group of 8 different enzymes that recognize the same sequence and cut in different locations

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9
Q

Alkaline phosphatase

A

An enzyme used for rDNA that will remove phosphate, and can help get rid of original vector by reducing clones

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10
Q

Klenow fragment

A

An enzyme used for rDNA that is used to convert an overhang to blunt end

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11
Q

Reverse Transcriptase & RNase H

A

An enzyme used for rDNA that is used for making cDNA

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12
Q

Taq DNA polymerase

A

An enzyme used for rDNA that is heat stable and used for PCR

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13
Q

Do you have to use the same enzyme for your gene of interest and expression vector?

A

YES

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14
Q

Transformation

A

A process in which one strain of bacteria is changed by a gene or genes from another strain of bacteria (ex. electroporation)

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15
Q

Screening

A

Evaluation of every protein for the desired property (ex. a growth condition where both mutant and wild type are able to grow but can be distinguished phenotypically)

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16
Q

Selection

A

Automatically eliminates nonfunctional variants (ex. a growth condition that allows for the selective propagation of genetically marked cells)

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17
Q

Screening & Selection

A

Two methods of library analysis

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18
Q

Functional Complementation

A

Procedure for screening a DNA library to identify the wild-type gene that restores the function of a defective gene in a particular mutant. (Results are original host cell, host cell + gene of interest, host cell + other genes)

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19
Q

Metabolic Load

A

The portion of a host cell’s resources that is required. to maintain and express foreign DNA, as either RNA or protein, in the cell (want to reduce)

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20
Q

Replication

A

3 major features:
1) Origin of Replication
2) Promoter
3) Selection Marker

(Think of temp.-selective vector ex.)

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21
Q

Manipulation of Expression (in Prok.)

A

1) Replication
2) Transcription
3) Translation
4) Stability/Fusion
5) Secretion

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22
Q

Transcription

A

Can be affected in multiple ways
1) Different Promoters
2) Different Temps.
3) Can be turned on/off
4) Repressor & Activators

(Think of lac operon)

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23
Q

Translation

A

Can enhance by overexpression of tRNA rate and can change the codons so host can recognize

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24
Q

Fusion

A

1) Marker Peptide for Immunoaffinity Column
2) Cleavage of Fusion (ex. Intein-mediated)
3) Phage Display for Screening of POI (Protein of Interest)
4) Fuse to Membrane of Host Cell (ex. color, antibiotic screening)

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25
Q

Factor Coexpression

A

Can overexpress some other factors to compensate for host cell (ex. chaperone proteins, DsbC, bacterial hemoglobin)

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26
Q

Specialized Host Cell

A

Limiting Biofilm (Biofilm-minus mutant): delete genes involved in pili, curli, colanic acid

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27
Q

Biofilm

A

Community of microorganisms living within a shared mass of secreted slime.

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28
Q

Genomic Intergration

A

1) Double Cross Over
2) Single Cross Over
3) 2 Step Selectable Marker
4) Removing Selectable Marker

reduces metabolic load

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29
Q

Double Cross Over

A
  • Clone gene of interest next to chromosomal DNA of
    host on both sides in plasmid (Preferred method)
  • Must be a noncritical sequence in host
  • Must be homologous to host cell’s site of recombination
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30
Q

Single Cross Over

A
  • Clone gene of interest next to chromosomal DNA of
    host on one side in plasmid
  • Only breaks in one place, entire plasmid gets into chromosome
  • Must be a noncritical sequence in host
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31
Q

2 Step Selectable Marker

A

Step 1) Insert marker gene into chromosomal DNA
Step 2) Insert target gene into chromosomal DNA
- screen until marker gene is gone, have to screen multiple times
- only use if there is no way you can monitor your gene of interest

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32
Q

Removing Selectable Marker

A

Marker gene + cloned gene are integrated into chromosomal DNA at same time, then marker is removed

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33
Q

Secretion

A
  • process increases protein folding
  • signal peptide doesn’t guarantee secretion
  • coexpress protein in host cell (protein will activate protein in host cell to help degrade cell membrane to get gene of interest secreted)
  • fuse w/ another system that gets secreted
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34
Q

Posttranslational Modification of Eukaryotic Proteins

A

proteolytic activation (to become active have to proteolytic cleavage)

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35
Q

O-linked oligosaccharides

A
  • sialic acid vs. mannose (how they affect protein expression)
  • o for oxygen (-OH Thr, Ser)
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36
Q

N-linked oligosaccharides

A
  • n for nitrogen (Asn)
  • has fucose
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37
Q

General Features of Eukaryotic Expression Systems

A

1) Origin of Replication
2) Promoter
3) Selection Marker (ESM for Euk.)

  • don’t need a cell wall
  • need E.coli origin of replication to make it easier to make more plasmid
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38
Q

Systems of Expression in Euk.

A

1) Fungus-Based Systems
2) Baculovirus-Insect Cell Systems
3) Mammalian Cell Systems

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39
Q

Fungus-Based Expression Systems

A

1) Single Cell Yeast
2) Filaments Fungi (multi cell)

  • when you change the expression vector you have to change the promoter
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40
Q

Single Cell Yeast

A

1) YEp (yeast episomal plasmid)
2) YIp (yeast integrating plasmid)
3) YAC (yeast artificial chromosome)

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41
Q

YEp

A
  • elevate product levels
42
Q

YIp

A
  • linear DNA is better for recomb.
  • reduce metabolic load
  • double cross over or single cross over approach
43
Q

YAC

A
  • have to have rep. origin, centromeres, telomeres
  • before entering host cell:
  • cut the gene of interest down the middle
  • insert DNA and ligate
  • increase chance of gene of interest getting in
44
Q

ARS

A

autonomous replicating sequence

45
Q

Filaments Fungi Advantages

A

1) grow on inexpensive media, secretion, mRNA processing
2) other posttranslational modifications (e.g., proteolysis)
3) in addition, mammalian-like glycosylation
4) nonhomologous-end-joining pathway

46
Q

Baculovirus-Insect Cell Systems

A
  • close to mammalian cell
  • use baculovirus as expression vector and it will infect insect cell (inject rDNA into insect cell)
  • ex.) in vitro recom.; tk produces an enzyme converting ganciclovir into a toxic product
  • ex.) N-glycosylation in humanized insect cells (from ER to Golgi)
47
Q

Mammalian Cell Systems

A
  • grow slower, complex nutritional needs but frequently this is the only choice for certain protein products
  • general features: SMG: selectable marker gene; pa: polyadenylation sequence (multiple cloning sites); a euk. replication origin usually from an animal virus
  • need E.coli replication origin + selection marker
  • ex.) signal S, tag T, cleavage site P, stop codon SC, and Kozak sequence K for translational control
48
Q

Two-vector expression system for Mammalian Cell Systems

A
  • have multiple subunits w/ different sequences
  • can’t separate
  • have to express each subunit inside host cell
  • have to have a way to select for each subunit (use different selection markers)
  • when made in the same cell, subunits then meetup
  • high metabolic load
49
Q

Two-gene expression vector for Mammalian Cell Systems

A
  • similar to two-vector expression system
  • only difference only one vector needed because genes are on the same vector
  • only need one selection marker
  • less metabolic load
50
Q

Bicistronic expression vector; IRES (internal ribosomal entry site) for Mammalian Cell Systems

A
  • make into single transcription unit (only one thing to transcribe)
  • need to insert IRES (similar to Kozak seq.)
  • even less metabolic load
  • only one expression vector
51
Q

Strategies to Increase Yield

A
  • cell death delay and higher cell density in bioreactors
  • induce stress
  • maximize protein production
  • add gene to bind to p53 to delay cell death
  • upregulating chaperones and secretion machinery
  • protein folding in the ER
  • overexpression of BiP (chaperone), PDI (isomerase), or Hac1 (TF that controls UPR(unfolded-protein response))
  • improves secretion
52
Q

Location of Recombinant Proteins

A

1) Medium (~ 10 proteins)
2) Periplasm (~ 100 proteins)
3) Cytoplasm (~ 2000 proteins)

53
Q

Sample Prep. Processes

A

Intracellular Expression:
- Insoluble in cytoplasm
* cell lysis + cell debris removal + harvest inclusion bodies + solubilization + purification
- Soluble in cytoplasm
* cell lysis + cell debris removal + recover supernatant + purification
- Periplasmic space
* cell wall disruption + cell removal + recover clarified sample + purification

Extracellular Expression:
- Culture medium
* cell removal + recover clarified sample + purification

54
Q

Inclusion Bodies

A

contains up to 90% purity
dark section is the bacteria

55
Q

Cell Breakage

A

Mechanical:
- Liquid Shear
1) Ultrasound
2) Mechanical agitation
ex.) Mickel blending sonomec
3) Pressure
ex.) French press
- Solid shear
1) Grinding
ex.) Pestel and mortar
2) Pressure
ex.) Hughes press and “X”-press

Nonmechanical:
- Desiccation
ex.) air, freeze, vacuum, and solvent drying
- Lysis
1) Physical
ex.) osmatic shock, pressure release, freeze + thaw
2) Chemical
ex.) antibiotics, cationic + anionic detergents
3) Enzymatic
ex.) Lysozyme + related enzymes, phage lysis, antibiotics

56
Q

Planning Considerations

A

1) define objectives
2) describe properties of target protein + critical impurities
3) develop analytical assays
4) minimize sample handling
5) minimize use of additives
6) remove damaging impieties early
7) use different methods at each step
8) minimize # of steps

57
Q

yield vs steps

A

less steps means higher yield

58
Q

Objectives

A
  • how pure (purity) or how much (quantity)?
  • how high activity?
  • different application calls for different amounts
  • how stable?
59
Q

Properties of the Target Protein and Impurities

A
  • properties of staring materials
    ex.) pH, stability, concentration, size, buffers
  • know what can affect downstream purification
  • know what it needs + what it doesn’t need
60
Q

Additives

A
  • know why you need them
  • know what they do
  • consider if they are necessary
  • ex.) detergents, reducing agents, protease inhibitors
61
Q

Zwitterions

A

a neutral mol. with + & - charges

62
Q

Sample Prep + CIPP

A
  • (Capture - Intermediate Purification - Polishing)
  • use of precipitation
  • precipitants
  • purification systems
  • purification table
  • separation principles
  • protein liquid chromatography
  • chromatography techniques: stationary and mobile phases
63
Q

3 Ways to Use Precipitation

A

1) clarification + supernatant + purification
2) extraction/clarification/concentration + redissolve pellet + purification
3) extraction/clarification + concentration + redissolve pellet + purification

64
Q

Purification Table

A
  • important to monitor purification progress (steps)
  • made based on your experiments
65
Q

Separation Principles

A

1) Size + Shape
2) Binding Site
3) Charged groups
4) Hydrophobic patches
5) Thiol groups
6) Metal chelating groups

66
Q

Matrix

A

chromatography matrices
- inorganic materials
ex.) porous silica
- synthetic organic materials
ex.) polyacrylamide
- polylactides
ex.) agarose, cellulose

67
Q

Microporous vs. Macroporous

A

microporous: polyacrylamide dextran: point crosslinked

macroporous: agarose

68
Q

Composite Gels

A

microporous polymers in macroporous pores

69
Q

Separation Parameters k and Rs

A
  • chromatogram
  • normalized retention factor, k
  • resolution Rs
70
Q

Separation of Peaks

A

more separation means more resolution

71
Q

Selectivity

A

distance between peaks
- more distance means more selective
- want Rs of 1.5 or better, if nit they overlap

72
Q

Efficiency

A

broadness of a peak
- thinner + taller peak means more efficient

73
Q

Factors Affect Rs

A
  • efficiency
  • tubing diameter
  • dirty medium
  • sample size
  • sample volume
74
Q

Factors of Efficiency

A
  • diffusion
    ex.) small bead size
  • longitudinal diffusion
    ex.) uniform packing, evenly distribute flow
75
Q

Effect of Tubing Diameter on Resolution

A
  • narrow tubing is better
  • wider tubing loses more efficiency
76
Q

Effect of Dirty Medium on Resolution

A
  • fresh medium is best
  • make sure to clean medium
  • binding capacity decreases as the medium gets more dirty
77
Q

Influence of Sample Size

A
  • smaller is better
  • if bigger the peaks may overlap
78
Q

Effect of Sample Volume on Resolution

A
  • volume increases so does the wideness of peaks
  • wider peaks means losing resolution
79
Q

Sample Viscosity

A
  • higher viscosity = higher pressure needed for the capillary system to keep flow rate the same
  • high pressure could break column
  • high pressure could cause gap + allow pressure to drop (lose efficiency)
  • temp. plays a factor
  • additives can increase viscosity of sample
80
Q

Charged Amino-Acid Side Chains

A
  • high pka = + charge
  • low pka = - charge
81
Q

Ion Exchange Chromatography (IEX)

A

separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids

82
Q

Ion Exchange Chromatography (IEX) Steps

A

1) Equilibration (low [ ] of salt)
2) Sample Application (what doesn’t bind, falls through)
3) Elution 1 (increase [ ] of salt)
4) Elution 2
5) Elution 3
6) Wash (put highest salt [ ] to get everything out)

83
Q

Selectivity and Buffer pH

A
  • selectivity is based on pH
  • want to maximize separation need to look at titration curve
84
Q

Protein Titration Curve

A
  • not smooth
  • surface charge will change gradually as pH of environment changes
  • pH is the x-axis
  • charge is the y-axis
  • pI is the point where charge is zero and a pH is any #
  • half is cation and other is anion
85
Q

Cation Exchanger

A
  • matrix is -
  • solutes (target protein) is +
  • exchanges cations
86
Q

Anion Exchanger

A
  • matrix is +
  • solutes (target protein) is -
  • exchanges anions
87
Q

Selectivity and Elution (gradient)

A
  • load sample then slowly increase salt [ ]
  • its a linear gradient
  • know when target protein falls out
88
Q

Selectivity and Elution (step)

A
  • load sample w/ low salt [ ]
  • increase salt [ ] to peaks from gradient
  • rapidly increases
  • not linear, looks like steps
89
Q

Components of Ion Exchange Media

A

1) High porosity (increase surface area)
2) Inert
3) High physical stability
4) High chemical stability
5) Uniform particle size
6) Polymer or agarose based matrices

90
Q

MonoBeads

A

most commonly used matrix

91
Q

Agarose

A
  • good compromise
  • macro-porous
  • arranged in bundles
92
Q

Functional Groups on Ion Exchangers

A
  • need functional group to produce charge
  • high pI = anion exchangers, need - charge
  • low pI = cation exchangers, need + charge
93
Q

Strong Ion Excahngers

A
  • refers to how it goes against charge
  • doesn’t refer to binding strength
  • no to low variation in a wide variety of pH
  • vertical line
94
Q

Weak Ion Exchangers

A
  • refers to how it goes against charge
  • doesn’t refer to binding strength
  • lots of variation in pH
  • no vertical line
95
Q

Strong vs Weak Ion Exchangers

A

Strong advantages:
- easier to replicate results
- no intermediate form
- sample loading capacity is maintained at high or low pH since no loss of charge

Weak advantages:
- can offer different selectivity

Weak disadvantage:
- could end up w/ non reproducible results

96
Q

Total Ionic Capacity

A

of charged functional groups per ml of medium, this is a fixed amount

97
Q

Available Capacity

A

actual amount of protein which can bind to an IEX medium under experimental conditions
- depends on properties of protein and experimental conditions

98
Q

Dynamic Capacity

A

the amount bound during defined conditions
- depends on properties of protein and experimental conditions

99
Q

Applications

A

CIPP

100
Q

Buffers: (non-)volatile

A
  • for ion exchangers
  • need appropriate pka
    -need to titrate w/ acid or base
  • be aware of pka and solution when choosing
101
Q

Buffers: Volatile

A

allows buffer to evaporate
- gets rid of extra purification step

Biotech (Recomb. Protein Production) (2 decks)

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