BIOTEKNOLOGI FARMASI

PRODUKSI PROTEIN REKOMBINAN

Putu Yustiantara
Karakter Protein
Rekombinan
 Protein Structures

• Proteins
– Are complex molecules built of chains of
amino acids
– Have specific molecular weights
– Have electrical charge that causes them to
interact with other molecules
• Hydrophilic – water loving
• Hydrophobic – water hating

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 Protein Structures
• Structural Arrangement – four levels
– Primary structure is the sequence in which amino
acids are linked together
– Secondary structure occurs when chains of amino
acids fold or twist at specific points forming new
shapes due to the formation of hydrogen bonds
between hydrophobic amino acids
• Most common shapes are alpha helices and beta sheets
– Alpha helix = amino acids form right handed spiral so hydrogen
bonds stabilize the structure linking an amino acid's nitrogen
atom to the oxygen atom of another amino acid
– Beta sheet = hydrogen bonds link nitrogen and oxygen atoms
forming sheets that are parallel (chains run in same direction or
anti-parallel (chains alternate in direction)

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 Protein Structures

• Structural Arrangement – four levels

– Tertiary structures = 3 dimensional
polypeptides and are formed when secondary
structures are cross linked
• Tertiary structure of protein determines its function
– Quaternary structures are unique, globular,
three-dimensional complexes built of several
polypeptides

• Why are proteins so fragile?

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 Protein Structures

© 2013 Pearson Education, Inc.

 Protein Structures
• Protein Folding
– The structure and function of a protein depends on
protein folding
– If protein is folded incorrectly, desired function of a
protein is lost and a misfolded protein can be
detrimental
– 1951: two regular structures were described
• Alpha helices and beta sheets
• Structures are fragile; hydrogen bonds are easily broken
• Incorrectly folded proteins can lead to diseases including
Alzheimers, cystic fibrosis, mad cow, forms of cancer and even
some heart attacks
• *** a challenge of biotechnology is to understand and control
the protein folding in the manufacturing process
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 Protein Structures

• Glycosylation – post-translational modification

wherein carbohydrate units are added to specific
locations on proteins
• More than 100 post-translational modifications
occur within eukaryotic cells
• This change can affect the protein's activity by
increasing:
– its solubility
– orient proteins into the membranes
– extend the active life of a molecule in an organism
© 2013 Pearson Education, Inc.
 Protein Structures

© 2013 Pearson Education, Inc.

Protein Production
 Protein Production
• Proteins are valuable, complex and
fragile products
– Production of proteins is a long and
painstaking process
– Two major phases used in producing proteins
• Upstream processing includes the actual
expression of the protein in the cell
• Downstream processing involves purification of the
protein and verification of the function; a stable
means of preserving the protein is also required
*** The choices made during upstream processing
can simplify downstream processing
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 Protein Production
• Protein Expression: Upstream
Processing
– Genetically engineered bacteria can be grown
in large scale fermenters (anaerobic) or
bioreactors (aerobic)
• Computers monitor air in bioreactors keeping
oxygen levels and temperature ideal for cell growth
• When phase of cell growth is highest- the bacteria
promoter must be activated to stimulate foreign
gene expression
– Must activate gene in recombinant organism after the
organism has completed synthesizing important natural
proteins needed for its metabolism
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© 2013 Pearson Education, Inc.
 Protein Production
Protein Expression: Upstream Processing

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Which host cell expression system?

• E. Coli
• Yeast
• Insect cells
• Mammalian cells
• Cell Free*
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 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.
Summary
 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)
Principle in recombinant protein expression

Bioinformatics Target identification and cloning

Protein purification and production

Protein expression test

Applications
Terimakasih