Protein engineering and recombinant protein expression

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Protein engineering and recombinant protein
expression
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Outline

• Why bother with recombinant fusion proteinor
  protein engineering?
• Principle in recombinant protein expression
• 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)

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Why bother with recombinant fusion proteinor
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)
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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.

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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

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Principle in recombinant protein expression
Bioinformatics

Target identification and cloning
Protein purification and production
Protein expression test
Applications

• Applications

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Things need to be considered for recombinant
protein expression

• 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)

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Which host cell expression system?

• E. Coli
• Yeast
• Insect cells
• Mammalian cells
• Cell free

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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.

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• BL21(DE3) strain
• lon and ompT proteases deficient
• Carries a lambda DE3 lysogen, the lacI gene and
  lacUV5-driven T7 RNA polymerase

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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
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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.

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Commonly used affinity tag system in recombinant
protein expression

• expression and purification of maltose-binding
  protein fusions. (provides a factor Xa cleavage
  site).
• 2. expression and purification of
  Glutathione-S-transferase 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.

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Affinity tags can be deWned as exogenous amino
acid (aa) sequences with a high aYnity for a
speciWc biological or chemical ligand.
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• 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.

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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.
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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 GATCCXXXXXXX
XXXXX 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
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Popular promoters for heterologous protein
expression in E. coli

• 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.
• 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.
• Hybrid promoters - Ptac and Ptrc. Induced by
  IPTG, a lot stronger than Plac and Ptrp.
• PBAD - induced by arabinose (Invitrogen)
• 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.

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Where to express the recombinant proteins?

• 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.

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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.

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OPTIMIZING TRANSCRIPTION OF THE CLONED GENE

• 1. genetic fusion to strong promoters
  (transcriptional fusion).
• increased gene dosage (utilize the genes
• 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.

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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.

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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

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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 ion-
• exchange 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.