How to make a recombinant protein
Protein-based therapeutics
There are 2 types of therapeutics:
Small molecule therapeutics
E.g. Aspirin
Recombinant protein therapeutics
i.e. Hormones
Protein based (recombinant protein therapeutics) include:
- hormones
- Cytokines (proteins i.e. growth factors secreted by immune cells that affect other cells
- vaccines
- monoclonal antibodies (forming a clone, which is derived asexually from a single individual or cell)
1/3 of recombinant protein therapeutics are produce in E.coli
Currently there are
- 140 protein-based therapeutics have been approved
- 500 on clinical trials
Characteristics of E.coli
预 From left to right, increasing in complexity of organism
预 A concentrated gel-like matrix in the space between the inner and outer cytoplasmic membrane called the periplasmic space,
which is only found in gram-negative bacteria.
预 Depending on the modification the protein undergoes depends on what host to use to grow the protein.
预 To study a kinase will need to use either a yeast or insect cell
预 Bacterial and human cells have the same number of genes but complexity differs due to posttranslational modification this
shows the importance of posttranslational modification.
预 Mammalian cells often fold protein more precisely
预 If require a soluble protein, use a lower temp than body temp (37 degrees Celsius) i.e. 16 degrees Celsius
/
Basic mechanism of cloning in E.coli
Cut DNA and vector
Paste DNA into vector (plasmid)
Transform DNA into bacteria
Select transformed bacteria- ensure plasmid contains antibiotic resistance
, Screen colonies
Step 1- DNA restriction enzymes
R1 is the strain of E.coli used
Eco referring to E.coli
/
EcoR1
Frequency of EcoR1 site (GAATTC) is 1 in 4^6
1 in 4000 bases
E.coli has 4.106 bases
Why doesn’t the sequence get cut in E.coli?
- old DNA gets methylated and becomes resistant to EcoR1
- new DNA will get cut
Why does E.coli have EcoR1?
-Any invading DNA not methylated will be susceptible to cleavage
-protect self from incoming DNA
Restriction factors restrict the growth of the virus in the human body
There are many restriction enzymes
Blunt ends are less common than sticky endsin biotechnology due to 2 reasons:
Low yield obtained when using DNA ligase to stick the 2 strands together.
/Inserting DNA insert in the opposite orientation
Where are restriction enzymes found?
Bacteria
Step 2 paste DNA
After cutting the DNA and the plasmid
Plasmid must contain origin of replication and ampicillian
Ampicillian is a synthetic version of penicillian that kills bacteria (antibiotics)
Having an ampilicillan resistance gene ensures once vector inserted into bacterial host it will not kill the bacteri cell
Not unidirectional cloning so either end of insert can be added
To get directional cloning , use 2 enzymes for each site
ORI is the site where DNA replication begins
There are many origin of replication in eukaryotic cells
/
Protein-based therapeutics
There are 2 types of therapeutics:
Small molecule therapeutics
E.g. Aspirin
Recombinant protein therapeutics
i.e. Hormones
Protein based (recombinant protein therapeutics) include:
- hormones
- Cytokines (proteins i.e. growth factors secreted by immune cells that affect other cells
- vaccines
- monoclonal antibodies (forming a clone, which is derived asexually from a single individual or cell)
1/3 of recombinant protein therapeutics are produce in E.coli
Currently there are
- 140 protein-based therapeutics have been approved
- 500 on clinical trials
Characteristics of E.coli
预 From left to right, increasing in complexity of organism
预 A concentrated gel-like matrix in the space between the inner and outer cytoplasmic membrane called the periplasmic space,
which is only found in gram-negative bacteria.
预 Depending on the modification the protein undergoes depends on what host to use to grow the protein.
预 To study a kinase will need to use either a yeast or insect cell
预 Bacterial and human cells have the same number of genes but complexity differs due to posttranslational modification this
shows the importance of posttranslational modification.
预 Mammalian cells often fold protein more precisely
预 If require a soluble protein, use a lower temp than body temp (37 degrees Celsius) i.e. 16 degrees Celsius
/
Basic mechanism of cloning in E.coli
Cut DNA and vector
Paste DNA into vector (plasmid)
Transform DNA into bacteria
Select transformed bacteria- ensure plasmid contains antibiotic resistance
, Screen colonies
Step 1- DNA restriction enzymes
R1 is the strain of E.coli used
Eco referring to E.coli
/
EcoR1
Frequency of EcoR1 site (GAATTC) is 1 in 4^6
1 in 4000 bases
E.coli has 4.106 bases
Why doesn’t the sequence get cut in E.coli?
- old DNA gets methylated and becomes resistant to EcoR1
- new DNA will get cut
Why does E.coli have EcoR1?
-Any invading DNA not methylated will be susceptible to cleavage
-protect self from incoming DNA
Restriction factors restrict the growth of the virus in the human body
There are many restriction enzymes
Blunt ends are less common than sticky endsin biotechnology due to 2 reasons:
Low yield obtained when using DNA ligase to stick the 2 strands together.
/Inserting DNA insert in the opposite orientation
Where are restriction enzymes found?
Bacteria
Step 2 paste DNA
After cutting the DNA and the plasmid
Plasmid must contain origin of replication and ampicillian
Ampicillian is a synthetic version of penicillian that kills bacteria (antibiotics)
Having an ampilicillan resistance gene ensures once vector inserted into bacterial host it will not kill the bacteri cell
Not unidirectional cloning so either end of insert can be added
To get directional cloning , use 2 enzymes for each site
ORI is the site where DNA replication begins
There are many origin of replication in eukaryotic cells
/
