Dit document omvat de samenvatting (info slides + lesnotities) van les 2 van het vak 'genome technology and applications'. Les 4 was gewoon een inleiding van NGS voor de taak. Hierbij moet je in groep een presentatie geven over een bepaalde NGS techniek.
Expession cloning
- You can do it in bacteria or eukaryote cells
o Cloning in bacteria you always need to do
- Different types
o You only want the RNA (e.g. as hybridisa?on probe)
§ RNA hybridises to DNA -> RNA is used for hybridisa?on probes
o You want to express a small amount of protein in vitro Add RNA to Xenopus oocytes
(typical frog eggs) or rabbit re?culocyte lysate
§ You add the RNA to the kit -> makes a liGle bit of protein
o You want to study the localisa?on of the protein in the cell
§ Label with GFP, see where the protein is
o You want to purify a large amount of protein
§ express in a bacterium or yeast, if possible
o You want to create a mammalian cell line for specific experiments
§ Create a stable cell line
In bacteria
- Bacteria are used most oOen
- Advantages
o Fast and simple to grow
o Easy to grow in large volumes
- Can be toxic for the cell
- Use of an inducible promoter
o T7 promoter
§ Not recognized by endogenous E. coli RNA polymerase
§ Use of a host cell with T7 RNA polymerase under lac promoter
§ Use of β-gal inducer IPTG, induces lac promoter
§ Grow culture without IPTG
§ Induce expression with IPTG, harvest quickl
- Very simple to grow them, grow really fast, also in large volumes
- pET vector
o Cloning site
o T7 promotor
§ Place where a RNA polymerase binds
§ You use a special E. coli where you have the T7 RNA polymerase present in the
bacterial chromosome.
§ The T7 RNA polymerase is behind the LacZ promotor and this promotor is inducible
by a certain compound, called IPTG. If you add IPTG to the medium -> compound is
inducible -> T7 RNA polymerase is made -> bind on the promotor -> it will make
RNA and proteins
§ You don’t have to do it this way. If you use a promotor of E. coli itself it will start
make the protein right away. But if the protein is a liGle bit toxic the cell won’t
grow anymore
- Limita?ons
o Post transla?on modifica?on is missing in bacteria
§ OOen necessary for biological ac?vity of protein
§ Not be biologically ac?ve -> not useful for you
, o Large proteins are difficult to synthesize for bacteria
o Incorrect or inefficient folding
§ Overexpression leads to inclusion bodies
• Insoluble aggregates of wrongly folded protein
Fusion proteins
- Protein is expressed as a fusion protein
- Can increase yield or improve solubility
- OOen the addi?on of an affinity tag -> helps to purify the protein
o GST tag: Glutathione-S-transferase
§ Small pep?de with high affinity for glutathione
§ Purifica?on using affinity chromatography with glutathione
o Poly HIS tag
§ 6 consecu?ve his?dine residues
§ Large affinity for nickel
§ Purifica?on using affinity chromatography with nicke
- Affinity chromatography -> in 1 stap you can get high purity protein
o You add proteins to a column -> protein will bound very specifically on the column, the
other proteins are washed away. Then you can release your protein from the column
and you have your column really in pure form.
o add affinity tag for what you have a column -> poly HIST tag = add 6 HIS -> large affinity
for nickel -> purifica?on using the affinity chromatography
- Vector with some of these characteris?cs (pGEX vector)
o Promotor: Ptac promotor = hybrid promotor
o Gluthione S transferase tag
o Mul?ple cloning site
o 3 versions of the plasmid -> differ in the reading frame
o Thrombin site to cleave off the protein, the GST tag
In eukaryotes
- More difficult than in bacteria
- Some?mes necessary:
o if posGransla?onal modifica?on is present
o If the protein is not efficiently expressed in bacteria (toxic, too large, …)
Double casse1e vector
- Two different recombinant proteins within same cell can be useful in certain instances (e.g.
insulin)
- Two mammalian expression vectors can be cotransfected into host cells each with different
selectable marker gene
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