Protein expression

Question 1

why might someone choose euk expression vs prok?

Answer 1

can get post-trans mods

Question 2

what are the feature vectors>

Answer 2

Basic features
• Origin of replication (ori) • Selectable marker
• Multiple cloning site

Special features
• For specific purposes such as – Mutagenesis
– Protein expression

Question 3

what are the feature expression vectors?

Answer 3

• Promoter
• Terminator
• Multiple cloning site
• Ribosome binding site
• Asequenceforfusionproteinor“tag”

Question 4

common oris and there copy #s?

Answer 4

ColE1 and pUC– 15-30 and 300 copies per cell

Question 5

Incompatibility:two different plasmids using the same ori cannot be maintained in the same cell. They interfere with each other’s replication

Answer 5

ya

Question 6

describe kanamycin MoA and subsequent resolvation

Answer 6

inhibits 30S ribosome; phopshorylated to kanamycin-P via neomycin phosphotransferase II

Question 7

compare inducible and repressible promoter

Answer 7

inducible–gene normally off; add inducer to induce gene expression
repressible–gene normally on; and repressor to prevent induction

Question 8

commonly used promoters and

Answer 8

• lacpromoter:sequencethatcontrols transcription of lac genes; blocked by a repressor LacI protein, but induced by IPTG (isopropylthiogalactoside)
• trp promoter: sequence that controls transcription of trp genes; blocked by tryptophan, but induced by 3-β-indoleacrylic acid–IAA binds Trp aporepressor, inactivates–>transcription
• tac promoter : a hybrid of lac and trp promoters; stronger than either, inducible by IPTG–binds inhibitor LacI
• λPL promoter: controls the transcription of the bacteriophage λ → strong
- Blocked by λcI protein
- temperature sensitive mutant λcI857, which is active at 30oC, but inactive at 42oC (promoter is induced)
• T7promoter
- promoter of bacteriophage T7 DNA-dependent RNA polymerase (RNAP) gene
- Requires T7 RNAP for expression

Question 9

purpose of protein fusion?

Answer 9

• Improvestabilityorsolubility
• Facilitate purification procedures
- GST fusion (glutathione-conj’d agarose)–GST v soluble; can also be used to bind column (pruification)
- His-tag (metal chelation chromatography)
• Provide sensitive means of detection - GFP
- HA-tag
• Conferspecificlocalization

Question 10

• GTScouldaffectthefunctionofthefusion;can be removed using a signal peptide for protease

• Factor Xa: cuts after Arg of Ile-Glu/Asp-Gly-Arg
• thrombin: cuts after Arg of Leu-Val-Pro-Arg-Gly-Ser

Answer 10

ya

Question 11

GFP fusion
• 236 amino acids and about 27 kDa
• Hasrevolutionizedhowwestudyprotein localization and functions in cells; GFP fusion proteins can be visualized directly
• VariousGFPvariantsallowmultipleproteinsto be observed simultaneously
• Also,antibodytoGFPallowsdetectionofthe fusion protein by Western blotting

Answer 11

ya

Question 12

short polypeptide tags–purify by fusing, peptides bind to Abs; good for high affinity

Answer 12

ya

Question 13

study slide 29

Answer 13

ya

Question 14

general steps of expression in bacteria

Answer 14

• Insert coding sequence into MCS
• Transform bacterial host & selection (take into account codon bias of species–solved by mutagenesis)
• (Induce) high level protein production
• Protein purification

Question 15

what factors influence levels of protein expression?

Answer 15

• Promoter strength: frequency of transcription initiation
• Plasmid copy number: # of templates
• Codonbias:availabilityoftRNAspeciesin host organism
• Protein stability and recovery
– Degradation of foreign proteins
– Accumulation as insoluble proteins in bacterial “inclusion bodies”

Question 16

large DNA frags can be cloned into yeast

Answer 16

ya

Question 17

Promoters for yeast expression
- Should be tightly regulated, thus allowing the expression of proteins toxic to the cell
- Should have a high induction ratio to background noise
- Several are used and the most widely used
is GAL1 promoter

Answer 17

ya

Question 18

describe gal yeast promoter/induction

Answer 18

galactose induces inducer via gal3–>inducer binds to gal 80; therefore, with no gal 80 binding to gal 4, gal 4 is free to activate GAL genes (is a transcription factor)

Question 19

markers for yeast expression?

Answer 19

Markers:Genesinvolvedinthebiosynthesisof amino acids are often used. Cell lines would carry the recessive mutant, while expression vectors would carry a wild type gene, allowing positive auxotrophic selection
- His3 - Leu2 - Trp1 - Ura3
Selection on a medium lacking the particular amino acid

Question 20

why might we express proteins in mammalian cells?

Answer 20

Reasons
• For mammalian genes, it is an environment closer to the native environment – fewer problems in expression
• Proteinsarefoldedintothenativeform
• Allow the correct post-translational modifications
• Proteins can be secreted, allowing isolation of proteins from the medium

Question 21

what are the components of a mammalian plasmid vector?

Answer 21

Plasmid vectors
• Origin of replication (ori)
• Selectablemarker
• Multiple cloning site
• Forstrongandproperexpression
- Promoter
- Terminator (polyadenylation signal) - Intron

Question 22

what are the oris in mammalian plasmids?

Answer 22

• SV40 ori: from simian virus 40, which causes lytic infection
- Very high copy number in transfected cells - Good for transient expression
- Low frequency of stable transfection
• BK or BPV ori: from human BK and bovine papilomas virus, which cause latent infections
- low to moderate copy
- Plasmid DNA using the ori is maintained in cells autonomously and stably for a long time
• EBV ori: from human Epstein-Barr virus
- Replicon is even more stable than BK and BPV ori

Question 23

selectble markers in mammalian expression vectors?

Answer 23

• Endogenous mutant genes as markers
- mutant causes a defect which can be compensated by the wild type gene
- Marker is recessive and can only be used in the mutant cell lines; thus limited use
• Dominant markers
- Confer a phenotype that is novel to the cell - Usually drug resistance

Question 24

what is geneticin?

Answer 24

an antibiotic which inhibits elongation of protein synth in both proks and euks

Question 25

promoters in euks?

Answer 25

Strong promoters for general expression
• Viralpromotersoftenconferverystrong expression in a wide range of cell types, and commonly used in mammalian expression vectors
- SV40 promoter: early promoter of simian virus 40, found in both monkeys and humans
- CMV promoter: human cytomegalovirus immediate early promoter
- RSV LTR promoter: Rous sarcoma virus (a retrovirus causing sarcoma in chicken) long terminal repeat promoter

Question 26

why is tet preferred over the lacI regulation?

Answer 26

because LacI inhibition requires high levels of IPTG which is toxic to mammalian cells

Question 27

study tet–56-61

Answer 27

ya

Question 28

describe the tet off system

Answer 28

fuse TetR (repressor) DNA binding domain to DNA activation domain of herpes simplex virus VP16 transactivator–results in tTA that binds to tetO (operator)–>activates gene expression; tet binds tTA, removes it from promoter, turns off gene expression

Question 29

describe the tet on system

Answer 29

tTA protein further modified–binds promoter in the presence of tetracyline–>turns on gene expression