Lecture 10/11 Flashcards
What is a recombinant protein?
Proteins derived from the expression of recombinant DNA within living cells
Why express and purify recombinant proteins?
• Large quantities required for research study in vitro
i.e. for structure determination using X‐ray crystallography or NMR (nuclear magnetic resonance) experiments
• For therapeutics and commercial applications
Recombinant protein expression
Bacterial expression systems: regulation using the lac operon
Insect expression systems
Yeast & other expression systems
Commercially available vectors
pET
pGEX
pBAC
Fusion proteins: affinity tags (for purification)
6x His tag
GST
MBP
REQUIREMENTS OF A BACTERIAL EXPRESSION SYSTEM
- Expression vector (plasmid)
Sequences for expression and regulation of expression - Host bacterial cells (often modified)
Important properties:
(i) Regulate expression
(ii) Provide suitable environment for expression and correct folding
REQUIREMENTS FOR HIGH LEVEL EXPRESSION
- A strong regulated promoter:
- able to make large numbers of mRNA transcripts
- able to produce mRNA transcripts when required
- Effective translation of proteins from mRNA transcripts
- Correct processing and folding of the translated proteins
Key features required for protein
overexpression in a bacterial
expression system
-Bacterial promoter (P)
-operator (O) sequences
-Polylinker with unique sites for several restriction endonucleases (i.e. cloning sites)
-Transcription termination sequence
-Selectable genetic marker (e.g. antibiotic resistance)
-ori
-Gene encoding repressor that binds O and regulates P
In addition, expression
vectors/plasmids also often have a gene that encodes an affinity tag for purification
What is a promoter for
allow efficient transcription of the inserted gene (drives transcription)
What is a polylinker/MCS
site of insertion for gene of interest
What is a operator
permits regulation through a specific repressor that will bind to it
steps in Producing a recombinant proteins in a Bacterial Expression System
Step 1: Insert gene into bacterial expression plasmid
Ligate ‘gene’ insert into bacterial expression plasmid (at MCS) using DNA ligase
>recombinant vector/plasmid
Step 2: Transform recombinant expression vector into host cells (E. coli)
Step 3: Select for transformed E. coli host cells using selectable antibiotic marker
(DNA sequencing to ensure correct clone)
Step 4: Express protein using inducible promoter
Step 5: Purify protein
Lac operon: collection of three genes required for lactose transport and metabolism in E.coli
• Adjacent structural genes: lacZ (β‐galactosidase), lacY (lactose permease) and lacA (thiogalactosidase transacetylase) • Allow for the effective digestion of lactose in vivo • Lactose permease (cytoplasmic membrane) transports lactose into cell • β‐galactosidase (cytoplasm) cleaves lactose into glucose and galactose
Control mechanism in vivo for lac repressor
• Lac repressor (regulatory protein) encoded by lacI gene
• lacI gene is constitutively expressed
• In absence of lactose, Lac repressor binds tightly to operator sequence
• Lac repressor interferes with binding of RNAP to promoter
> prevents gene transcription by RNA polymerase
How to optimise translation
Need effective translation of proteins from mRNA transcripts
• Not all 61 mRNA codons are used equally
• Frequently used codons have large pools of their corresponding tRNAs
• The expression system generates large numbers of the target gene mRNA
What may go wrong in translation
codon usage in the over‐expressed protein differs significantly from the host cell, problems may arise during protein expression.
- truncated proteins
- mis-incorporation of amino acids (e.g. Lys for ARg at the AGA codon)
-inhibition of cell growth/protein synthesis
the expression system needs to maximise the translation of proteins from this mRNA
How to make sure that the expression system is maximising the translation of the protein form the corrrect mRNA
Use host strain that expresses genes encoding tRNAs for rare codons. A modified cells
E.coli BL21 strain (DE3) Rosetta : extra copies of genes encoding tRNA
example: Yield of human plasminogen activator protein was increased 10‐fold in E. coli Rosetta BL21
(DE3) strain due to an extra gene encoding tRNA for AGG and AGA (Arg)
What problem may face during processing
- Many proteins require the formation of disulphide (S‐S) bonds to fold properly
- Without S‐S bond, proteins may be degraded or precipitate in the cell
- Expression of proteins occurs in cytoplasm of E. coli
- Cytoplasm is a reducing environment that can prevent S‐S bond formation
What is the solution to disulphide bonds not folding properly
Solution 1: Use E. coli strains engineered with mutations in:
• gor (glutathione reductase)
• trxB (thioredoxin reductase)
These altered E. coli strains can promote disulphide bond formation and correct folding in the cytoplasm
Solution 2: Export proteins to this compartment using a leader sequence to promote S‐S bond formation Leader sequence must be incorporated in the expression vector
What is an example of leader sequence for solution 2
PelB gene:
encodes periplasmic leader sequence of 22 amino acid residues. Localises to periplasm
What is the pET vector
It contains a expression feature that increases the control on protein induction. An extra level of protein induction. Pt7: promoter for T7 RNA polymerase. But a genetically modified host cell is needed to contain the T7 RNA polymerase gene as well as its normal indulge-nous polymerase gene (eg of host cell is BL(DE3) Host chromosome
How does the IPTG impact a plasmid
Under normal growth: • System is repressed by Lac repressor protein which is transcribed from the lacI gene by host cell RNA polymerase induction of IPTG
• Lac repressor inactivated on both plasmid and bacterial genomic DNA
• T7 RNA pol is produced by host cell RNA pol (under control of Plac promoter, transcribed from host
DE3 lysogen)
• T7 RNA Polymerase can now transcribe the gene of interest (target gene, downstream of PT7
promoter on pET vector)
over‐expression of protein from target gene
What is another commercially available vector
pGEX, Ptac modified promoter to increase binding of RNA polymerase
• System is
repressed by lac repressor
Add IPTG
• Endogenous E. coli RNA polymerase will bind promoter and transcribe downstream genes
• Proteins will be translated using the host cells translation machinery
How can the structure of the domain help to facilitate the purification process
• Proteins have structural domains: fold independently and have discrete
functions
Can add domains to proteins that can be used to facilitate solubility, folding and enhance purification
The added domains are call affinity tag
What are the 3 affinity tags
6xHis-tag, interacts with cobalt and nickel ions, MBP (Maltose-binding protein), interacts with maltose (amylose), GST (glutathione-S-transferase) interacts with reduced glutathione.
REMOVAL OF AFFINITY TAGS BY PROTEASE DIGESTION
- Protease cleavage sites must be encoded when preparing constructs (expression vector) for expressing proteins
- Must ensure the recognition site is not present in the expressed protein
What are the commonly used proteases for removal of affinity tags
Thombin(Thr) that cleaves LVPR/GS
Factor Xa that cleaves I(D)EGR
Enterokinase that cleaves EEEEK
3C human rhino virus protease(preScission) LDVLFQ/GP
Properties of Polyhistidine moiety (6x Histidine‐tag)
• Rarely effects protein structure
• Binds to a nickel (Ni) column under both native and denaturing conditions
IMAC: Immobilised Metal Affinity Chromatography
• Uses agarose or magnetic beads as supports
• Metal chelators are ligands (e.g. Ni‐nitrilotriacetic acid) to which His‐tag binds
• Wash column extensively
• Elute with [high] imidazole
How does the affinity tag Polyhistidine moiety (6x Histidine‐tag) work in steps
- Induce expression of fusion protein with a 6x His‐Tag (cell‐free extract)
- Bind protein to Ni‐NTA column (can occur under native or denaturing conditions)
- Wash to ensure removal of unbound/non-specific bound proteins
-amino acid analogue
-competes with the polyhistidine tag for binding to the Ni-NTA column
4A. Elite the His-tagged protein with imidazole ([Hight]) - Add protease to cleave His-tag from recombinant protein
4B. When protein is still bound at column, add protease to release
Properties of Glutathione S‐transferase (GST) 26 kDa
- Fusion protein with GST tag binds reduced glutathione (GSH) attached to a mattrix (e.g. agarose)
- GSH is both the substrate for GST and is used to elute proteins from the GST colume (note: in excess)
- GST-fusion maintains solubility of proteins
