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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
Factor Coexpression
Can overexpress some other factors to compensate for host cell (ex. chaperone proteins, DsbC, bacterial hemoglobin)
26
Specialized Host Cell
Limiting Biofilm (Biofilm-minus mutant): delete genes involved in pili, curli, colanic acid
27
Biofilm
Community of microorganisms living within a shared mass of secreted slime.
28
Genomic Intergration
1) Double Cross Over 2) Single Cross Over 3) 2 Step Selectable Marker 4) Removing Selectable Marker reduces metabolic load
29
Double Cross Over
- 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
30
Single Cross Over
- 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
31
2 Step Selectable Marker
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
32
Removing Selectable Marker
Marker gene + cloned gene are integrated into chromosomal DNA at same time, then marker is removed
33
Secretion
- 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
34
Posttranslational Modification of Eukaryotic Proteins
proteolytic activation (to become active have to proteolytic cleavage)
35
O-linked oligosaccharides
- sialic acid vs. mannose (how they affect protein expression) - o for oxygen (-OH Thr, Ser)
36
N-linked oligosaccharides
- n for nitrogen (Asn) - has fucose
37
General Features of Eukaryotic Expression Systems
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
38
Systems of Expression in Euk.
1) Fungus-Based Systems 2) Baculovirus-Insect Cell Systems 3) Mammalian Cell Systems
39
Fungus-Based Expression Systems
1) Single Cell Yeast 2) Filaments Fungi (multi cell) - when you change the expression vector you have to change the promoter
40
Single Cell Yeast
1) YEp (yeast episomal plasmid) 2) YIp (yeast integrating plasmid) 3) YAC (yeast artificial chromosome)
41
YEp
- elevate product levels
42
YIp
- linear DNA is better for recomb. - reduce metabolic load - double cross over or single cross over approach
43
YAC
- 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
ARS
autonomous replicating sequence
45
Filaments Fungi Advantages
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
Baculovirus-Insect Cell Systems
- 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
Mammalian Cell Systems
- 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
Two-vector expression system for Mammalian Cell Systems
- 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
Two-gene expression vector for Mammalian Cell Systems
- 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
Bicistronic expression vector; IRES (internal ribosomal entry site) for Mammalian Cell Systems
- 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
Strategies to Increase Yield
- 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
Location of Recombinant Proteins
1) Medium (~ 10 proteins) 2) Periplasm (~ 100 proteins) 3) Cytoplasm (~ 2000 proteins)
53
Sample Prep. Processes
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
Inclusion Bodies
contains up to 90% purity dark section is the bacteria
55
Cell Breakage
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
Planning Considerations
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
yield vs steps
less steps means higher yield
58
Objectives
- how pure (purity) or how much (quantity)? - how high activity? - different application calls for different amounts - how stable?
59
Properties of the Target Protein and Impurities
- 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
Additives
- know why you need them - know what they do - consider if they are necessary - ex.) detergents, reducing agents, protease inhibitors
61
Zwitterions
a neutral mol. with + & - charges
62
Sample Prep + CIPP
- (Capture - Intermediate Purification - Polishing) - use of precipitation - precipitants - purification systems - purification table - separation principles - protein liquid chromatography - chromatography techniques: stationary and mobile phases
63
3 Ways to Use Precipitation
1) clarification + supernatant + purification 2) extraction/clarification/concentration + redissolve pellet + purification 3) extraction/clarification + concentration + redissolve pellet + purification
64
Purification Table
- important to monitor purification progress (steps) - made based on your experiments
65
Separation Principles
1) Size + Shape 2) Binding Site 3) Charged groups 4) Hydrophobic patches 5) Thiol groups 6) Metal chelating groups
66
Matrix
chromatography matrices - inorganic materials ex.) porous silica - synthetic organic materials ex.) polyacrylamide - polylactides ex.) agarose, cellulose
67
Microporous vs. Macroporous
microporous: polyacrylamide dextran: point crosslinked macroporous: agarose
68
Composite Gels
microporous polymers in macroporous pores
69
Separation Parameters k and Rs
- chromatogram - normalized retention factor, k - resolution Rs
70
Separation of Peaks
more separation means more resolution
71
Selectivity
distance between peaks - more distance means more selective - want Rs of 1.5 or better, if nit they overlap
72
Efficiency
broadness of a peak - thinner + taller peak means more efficient
73
Factors Affect Rs
- efficiency - tubing diameter - dirty medium - sample size - sample volume
74
Factors of Efficiency
- diffusion ex.) small bead size - longitudinal diffusion ex.) uniform packing, evenly distribute flow
75
Effect of Tubing Diameter on Resolution
- narrow tubing is better - wider tubing loses more efficiency
76
Effect of Dirty Medium on Resolution
- fresh medium is best - make sure to clean medium - binding capacity decreases as the medium gets more dirty
77
Influence of Sample Size
- smaller is better - if bigger the peaks may overlap
78
Effect of Sample Volume on Resolution
- volume increases so does the wideness of peaks - wider peaks means losing resolution
79
Sample Viscosity
- 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
Charged Amino-Acid Side Chains
- high pka = + charge - low pka = - charge
81
Ion Exchange Chromatography (IEX)
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
Ion Exchange Chromatography (IEX) Steps
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
Selectivity and Buffer pH
- selectivity is based on pH - want to maximize separation need to look at titration curve
84
Protein Titration Curve
- 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
Cation Exchanger
- matrix is - - solutes (target protein) is + - exchanges cations
86
Anion Exchanger
- matrix is + - solutes (target protein) is - - exchanges anions
87
Selectivity and Elution (gradient)
- load sample then slowly increase salt [ ] - its a linear gradient - know when target protein falls out
88
Selectivity and Elution (step)
- load sample w/ low salt [ ] - increase salt [ ] to peaks from gradient - rapidly increases - not linear, looks like steps
89
Components of Ion Exchange Media
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
MonoBeads
most commonly used matrix
91
Agarose
- good compromise - macro-porous - arranged in bundles
92
Functional Groups on Ion Exchangers
- need functional group to produce charge - high pI = anion exchangers, need - charge - low pI = cation exchangers, need + charge
93
Strong Ion Excahngers
- 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
Weak Ion Exchangers
- refers to how it goes against charge - doesn't refer to binding strength - lots of variation in pH - no vertical line
95
Strong vs Weak Ion Exchangers
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
Total Ionic Capacity
of charged functional groups per ml of medium, this is a fixed amount
97
Available Capacity
actual amount of protein which can bind to an IEX medium under experimental conditions - depends on properties of protein and experimental conditions
98
Dynamic Capacity
the amount bound during defined conditions - depends on properties of protein and experimental conditions
99
Applications
CIPP
100
Buffers: (non-)volatile
- for ion exchangers - need appropriate pka -need to titrate w/ acid or base - be aware of pka and solution when choosing
101
Buffers: Volatile
allows buffer to evaporate - gets rid of extra purification step

Biotech (Recomb. Protein Production) (2 decks)

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