Transcript Lecture 1 III. recombinant protein

Prof. FATCHIYAH, M.Kes.PhD
http://fatchiyah.lecture.ub.ac.id
PROTEIN ENGINEERING AND
RECOMBINANT PROTEIN
EXPRESSION
Outline
1. Why bother with recombinant fusion protein
or protein engineering?
2. Principle in recombinant protein expression
3. Things need to be considered for recombinant
protein expression
a. How to produce?
b. How to make an expression recombinant DNA
construct?
c. Where to express?
d. Difficulties (protein expression problems)
Why bother with recombinant fusion protein
or protein engineering?
1. to minimize proteolysis.
2. for efficient and selective purification.
3. to optimize translation efficiency.
4. for different applications
(specific expression scenarios): antibody
production, biochemical experiments,
structural biology, industrial usage.
(protein biotechnology or protein engineering)
Protein biotechnology or engineering
Definition:
Deliberate design and production of proteins with
novel or altered structure and properties, that are not
found in natural proteins.
• To study protein structure and function
• Applications in industry (enzymes) and medicine (drugs)
-- New and improved proteins are always wanted.
Example: Extremophilic proteins have been found in nature
(temperatures, salt concentrations, pH values) could be useful.
Applications
Functional Studies
Enzymatic Assays
Protein-protein interactions
Protein Ligand Interactions
Structural Studies
Protein Crystallography & NMR Structure Determination
Target Proteins for Rational Drug Design
Therapeutic Proteins – Preclinical Studies
Principle in recombinant protein expression
Bioinformatics
Protein purification and production
Applications
• Applications
Target identification and cloning
Protein expression test
Things need to be considered for recombinant
protein expression
1. How to produce?
choose for protein expression system (vector and
host)
2. How to make an expression recombinant DNA
construct?
translational or transcriptional fusion, promoter use
(inducible or constitutive)
3.
Where to express?
cytosol, periplasm, secretion, inclusion body
4. Difficulties (protein expression problems)
Which host cell expression system?
• E. Coli
• Yeast
• Insect cells
• Mammalian cells
• Cell free
Choose of protein expression system
The KEY idea is the cloned gene must be transcribed and
translated most efficiently.
Expression vector: MAXIMIZE GENE EXPRESSION.
Host: MINIMIZE TURNOVER OF GENE PRODUCTS
(preventing proteolysis in vivo in E. coli).
---- Use protease deficient mutants as hosts.
Lon - a major ATP-dependent protease in E. coli. Has
broad specificity for unfolded or misfolded
proteins in vivo. lon mutants - pleiotropic, but
two main phenotypes - mucoidy and UV
sensitivity.
ompT - an outer membrane localized protease. Cleaves
at paired basic residues.
degP - periplasmic protease - could inactivate some
secreted proteins.
• BL21(DE3) strain
– lon and ompT proteases deficient
– Carries a lambda DE3 lysogen, the lacI gene and
lacUV5-driven T7 RNA polymerase
Increase selectivity of
protein purification:
(Gene fusion strategies)
Most target protein lack a suitable
Affinity ligand usable for capture on
a solid matrix. A way to circumvent this
obstacle is to genetically fuse the gene
encoding the target protein with a gene
encoding a purification tag. When the
chimeric protein is expressed, the tag
allows for specific capture of the fusion
protein. This will allow the purification
of virtually any protein without any prior
knowledge of its biochemical properties.
Hearn and Acosta, 2001
Advantages and disadvantages for
using tags in fusion proteins
Plus factors:
(1)improve protein yield (2) prevent proteolysis (3)
facilitate protein refolding (4) protect the antigenicity of
the fusion protein and (5) increase solubility (6) increase
the sensitivity of binding assays for tagged ScFv.
Minus factors:
(1) a change in protein conformation (solubility and
activity) (2) lower protein yields (cleavage may not be
complete) (3) inhibition of enzyme activity (4) alteration
in biological activity (5) undesired flexibility in structural
studies (6) cleavage/removing the fusion partner requires
expensive protease (Factor Xa, enterokinase) and (7)
toxicity.
Commonly used affinity tag system in
recombinant protein expression:
1. expression and purification of maltose-binding
protein fusions. (provides a factor Xa cleavage site).
2. expression and purification of Glutathione-Stransferase fusion proteins. (contains either a
thrombin cleavage site, a factor Xa cleavage site, or
an Asp-Pro acid cleavage site).
3. expression and purification of thioredoxin fusion
proteins. (provides an enterokinase cleavage site).
4. expression and purification of 6X His-tagged
proteins.
Affinity tags can be deWned as exogenous amino acid (aa) sequences with
a high aYnity for a speciWc biological or chemical ligand.
Transcription vectors:
1. Vector itself already contains its own promoter and terminator
sequences for efficient transcription initiation and termination.
Therefore, No potential translation initiation site ahead of the
cloning site should be provided by incoming cloned DNA.
2. Transcriptional fusion is a gene construct that investigates
transcription activity of a gene of interest.
Translation vectors:
1. Vector itself contains a segment from a specific gene whose
protein product is synthesized more rapidly than any other
protein during transformation or infection. Target DNA is fused to
either 2nd or 11thcodon of gene 10.
2. The translational fusion bears the promoter of your gene and other
sequence surrounding it (C-terminal) as well as the N-terminal sequence
of your gene. The reporter gene is then inserted between these two
terminals and in-frame such that you have one long protein product.
Architecture of reporter gene constructs
(A) Transcriptional reporter, (B) translational reporter
Transcriptional reporters consist of a promoter fragment from a gene of
interest driving GFP (Figure 1A). Typically, promoter fragments of a few
kilobases immediately upstream of the start codon contain a significant
portion of the cis-regulatory information necessary to provide a tentative
expression pattern of the endogenous gene under study.
Translational reporters are in-frame gene fusions between GFP and a gene of
interest (Figure 1B). Ideally, a translational reporter includes the entire
genomic locus of a gene (5’ upstream region, exons, introns, 3 UTR). GFP can
be inserted at any point in the open reading frame, preferably at a site that
does not disrupt protein function or topology.
Translational fusion
Assume the restriction site identified in the gene which you want to express
is a BamHI site. Digest with BamHI to obtain:
GATCCXXXXXXXXXXXX
GYYYYYYYYYYYYY
↓
Treat with Klenow fragment to fill in the unpaired bases to obtain:
GATCCXXXXXXXXXXXX
CTAGYYYYYYYYYYYYY
↓
Determine the proper reading frame of the gene. Assume the coding sequence
of the filled-in fragment should read:
GA TCC XXX XXX XXX
↓
Determine which restriction endonuclease should be used to digest
an expression vector pSKF301 in order to allow expression of the fusion protein.
For this example, StuI is required to yield:
ccatg gat cat atg tta aca gat atc aag gGA TCC XXX XXX
pSKF301 (carrier sequence)
your fusion gene
Popular promoters for heterologous
protein expression in E. coli
1. Plac. Negatively regulated by lacI. Need for sufficient levels of repressor (lacIq
and lacIq1 alleles on vectors). PlacUV5 is very popular because its regulation is
not dependent on CAP.
2. Ptrp. Negatively regulated by trpR. Vectors containing this promoter can be
transformed into any strain, easy induction by starvation for tryptophan. Not
suitable for expression of proteins with high Trp content.
3. Hybrid promoters - Ptac and Ptrc. Induced by IPTG, a lot stronger than Plac
and Ptrp.
4. PBAD - induced by arabinose (Invitrogen)
5. T7 system. Uses T7 promoters, which require T7 RNA polymerase. T7 RNA
polymerase (encoded by T7 gene 1) has stringent specificity for its own
promoters. It initiates and elongates chains 5 times faster than E. coli RNA Pol
and is resistant to Rifampicin (unlike E. coli Pol).
6. pET series of vectors (Rosenberg et al, 1987, Gene).
pET - Plasmid for Expression by T7 RNA pol. Commercially available by
Novagen.
Where to express the recombinant proteins?
1. Direct expression (cytosol): E. coli cytoplasm is a reducing
environment - difficult to ensure proper disulphide bonds
formation.
2. Fusion expression (inclusion body?): Ensures good
translation initiation. Can overcome insolubility and/or
instability problems with small peptides. Has purification
advantages based on affinity chromatography.
3. Secretion (periplasm or medium): a fusion alternative when
proteins are fused to peptides or proteins targeted for
secretion. Periplasm offers a more oxidizing environment,
where proteins tend to fold better. Major drawbacks: limited
capacity for secretion (0.1-0.2% total cell protein compared to
10% produced intracellularly) and inability for
posttranslational modifications of proteins.
General problems with heterologus gene
expression
(a) Not enough protein is produced:
* codon usage preferential (rare codon)
* potential mRNA secondary structure.(5’-end
ATcontent, 3’-end transcriptional terminator)
* toxic gene.
(b) Enough protein is produced, but it is insoluble:
* vary the growth temperature.
* change fermentation medium.
* low-copy-number plasmas.
* selection of promoter.
The KEY idea is to slow down the expression rate of
protein.
OPTIMIZING TRANSCRIPTION OF THE
CLONED GENE
1. genetic fusion to strong promoters
(transcriptional fusion).
2. increased gene dosage (utilize the gene’s
own promoter with the gene on a high-copy
plasmid).
3. potential problem with toxic genes and
available methods for efficient repression.
4. solutions to potential problems with
premature termination and mRNA instability.
OPTIMIZING TRANSLATION OF THE
CLONED GENE
1. sequence determinants for translation
initiation (Shine-Delargo sequence).
2. translational fusion vectors.
3. potential problem with biased codon
usage.
4. enhancing the stability of protein
products.
Insolubility of heterologous proteins
produced in E.coli
Inclusion bodies.
Dense particles, containing precipitated proteins.
Their formation depends on protein synthesis
rate, growth conditions.
Advantages: proteolysis resistant, big yield,
relatively pure, easy to separate.
Disadvantages: inactive product requires in
vitro refolding and renaturation
Refolding of recombinant proteins
Solubilisation:
High T 0 C, detergents, high concentration of inorganic salts or organic
solvents all used. The most commonly used organic solutes such as
urea or guanidine-HCl often used in the presence of reducing agents
(mercaptoethanol or DTT). Solubilized proteins can be purified by ionexchange chromatography or other conventional methods, prior to
refolding.
Refolding:
If no S-S bonds present - remove denaturing agent to allow protein to
fold correctly. If S-S bonds present - their formation can be
accomplished: by air oxidation, catalysed by trace metal ions; by a
mixture of reduced and oxidized thiol compounds - oxidized DTT,
reduced DTT; GSSG/GSH; cystine and cysteine, cystamine and
cysteamine.