3.

Production of
Recombinant Proteins
What is recombinant protein?
Recombinant proteins are proteins encoded by
recombinant DNA that has been cloned in an expression
vector that supports expression of the gene and translation
of messenger RNA.

recombinant DNA : bring together genetic materials

from multiple source, creating sequences that would not
other with be found in the genome
What are recombinant proteins used for?
(1) Biomedical research to understand health and disease
(e.g. antibody, understanding protein-protein interaction etc.)
(2) Biotherapeutics
(e.g. vaccine, insulin, hormone etc.)
• Protein Production in Prokaryotic Hosts

• Heterologous Protein Production in

Eukaryotic Cells

• Protein Engineering
 Protein Production in
Prokaryotic Hosts
q Central Dogma
 Protein Production in
Prokaryotic Hosts
q Regulating transcription
What is minimal requirement for an effective gene
expression system?
§ Promoter (strong, weak, regulatable…)
§ What is strong promoter? One that has high affinity
for RNA polymerase
§ Regulatable promoter? (inducible promoter)

A strong E.coli promoter resemble the consensus sequences

for the -35 and -10 boxes that bind to RNA polymerase
 Protein Production in
Prokaryotic Hosts
q Regulating transcription

1. Why do we need an inducible promoter for

recombinant protein production?
§ Factors that may increase the metabolic
burden on a prokaryotic host cells

2. What other inducers could be used as…

 Protein Production in
Prokaryotic Hosts
§ Lac Promoter
- The effects of the concentration of glucose and lactose in the
growth medium on the level of transcription from the E.coli lac
promoter.
 Protein Production in
Prokaryotic Hosts
§ Inducers of Lac promoter

Beta 1-4 linkage

synthetic inducer

Beta 1-6 linkage

inducer
 Protein Production in
Prokaryotic Hosts
§ T7 expression system
- pT7: promotor from gene10 of the E.coli bacteriophage T7
- T7 promotor is not recognized by the E.coli RNA polymerase, but
rather, it requires the T7 RNA polymerase for transcription
 Protein Production in
Prokaryotic Hosts
q Increasing Translation Efficiency
§ Ribosome-Binding Site (RBS)
- A translational initiation signal
- 6-8 nucleotide (e.g., UAAGGAGG) in mRNA that can base pair with a
complementary sequence on the 16S RNA component of the small
ribosomal subunit
- the ribosome-binding sequence must be located within a short distance
(usually 2 to 20 nt) from the translational start codon of the cloned gene
 Protein Production in
Prokaryotic Hosts
§ The expression vector : pET

1. Transcription
-Promotor (regulatable?)
-Operator
-Terminator
2. Translation
-Ribosome binding site
-Start codon
-Stop codon

Origin
Repressor
Selection marker

Then, where is the T7 RNA polymerase?

 Protein Production in
§ Optimizing Codon Usage Prokaryotic Hosts

- While genetic code is universal among organism, the codons that specify
amino acids are used to different extents in various organisms

- When a cloned gene has codons that are rarely used by the host cell, a
cellular incompatibility occurs that decreases translation efficiency

- Codon optimization could improve the gene expression

Genetic code and codon usage in E.coli and humans

 Protein Production in
Prokaryotic Hosts

-Alternatively, host cell that have been engineered to overexpress rare

tRNA are commercially available.

pRARE: encode 10 rare tRNAs

Genes encode 6 rare tRNAs integrated

into chromosome of E.coli
 Protein Production in
§ Increasing Protein Stability Prokaryotic Hosts

Ø Molecular chaperones

- High levels of expression of some foreign

proteins in bacterial hosts often results in the
formation of insoluble, inactive protein
aggregates known as inclusion bodies

- Chaperon is the molecule that helps protein

folding

E.coli proteins that facilitate the correct

folding of recombinant protein
 Protein Production in
Prokaryotic Hosts
Ø Fusion Protein

- The fusion partner may have intrinsic

chaperone activity attract chaperones, or be
highly acidic, thereby inhibiting protein
aggregation through electrostatic repulsions.
 Protein Production in
§ Increasing Protein Secretion Prokaryotic Hosts

- Directing a foreign protein to the periplasm or the growth medium

makes its purification easier and less costly, as many fewer
proteins are present outside the cytoplasmic membrane than in the
cytoplasm.
 Protein Production in
Prokaryotic Hosts

Ø Secretion via the Sec pathway (Type II)

Gram - Gram +
 Protein Production in
Prokaryotic Hosts

- Strategy to increase secretion of a recombinant protein to

the periplasm
 Protein Production in
Prokaryotic Hosts
Ø Secretion via the Type III secretion system
(Type III secretion systems are found in pathogenic bacteria)

- To export recombinant proteins via the T3SS, the target protein is fused
to the N-terminal sequence from the SptP protein that is naturally
secreted through this system
 Protein Production in
Prokaryotic Hosts

- Build a minimal T3SS system in nonpathogenic host

 Protein Production in
§ Facilitating Protein Purification Prokaryotic Hosts

Ø Affinity purification system

 Protein Production in
Prokaryotic Hosts

Ø Proteolytic Removal of Purification Tags

- Blood coagulation protein factor Xa

- Xa cleaves peptide bonds uniquely on the C-terminal side of the Ile-Glu-
Gly-Arg sequences
-Additional purification step is required to separate both the protease and
the cleaved peptide from the target protein
 Protein Production in
Prokaryotic Hosts
Ø Self-cleaving Purification Tags
- To avoid additional purification steps that are costly and may reduce
yields of target protein, self-cleaving purification tags can be used

- Intein (interventing protein) is an amino acid segment found in some

natural proteins that can excise itself and join the flaking segments
(exteins) with a peptide bond.

Self splicing proteins

 Protein Production in
Prokaryotic Hosts

- Modified intein can be used in removal of protein purification

tags