Summery applied biotechnology
1
, H1: peptides, proteins and enzymes
1. Biologicals and biotechnology – drugs
How do they work?:
- Activate (agonist) or deactivate (antagonist) an enzyme: binding
directly to the active site or through allosteric binding
- Add or remove a metabolite (or analogue)
- Add or remove a protein
- Activate, change or deactivate a biological response (cytokines,
steroids, peptides, gases)
- Alter the biological system temporarily or permanently
Where do they work?:
Drugs work in on receptors on the surface of the cell.
è An oral drug goes through your stomach, intestine, blood and it starts spreading in circulating system.
Once it survives the enzymes from the mouth, the low pH in the stomach and the intestines it goes to
the liver, where it will continue to go kidney until it gets eliminated.
è For small molecules there is easy penetration, but biological molecules get bigger (peptides, antibodies,
viruses, bacteria, human cells … as drugs) and have it more difficult to pass the membrane.
Different types of drugs have a different time of working. It depends on how they come in the body and how
they are eliminated. Some molecules go through you (they will never go into your blood system) and you will
just excrete them again. Antibodies can stay 20-28 days in your body. This cannot be injected orally, but you
deliver them intravenously. For gene therapy, one injection in your lifetime suffices.
Differences between chemical vs biological drugs:
Biopharmaceutical = an active drug substance made by a living organism or from a chemical reaction involving biological
parts.
è By biotherapeutic you never get 100% of the same molecules, this is different from chemics. You rely on the cell
that makes the product for you. This is a long-complicated process with different steps. All these different steps
can affect your final product.
2
, 2. Human growth hormone
hGH (a.k.a somatotropin) is made in the pituitary gland (= hypophysis) and
released in the blood stream.
è It has a direct effect on many cell types and activates IGF-1
synthesis in the liver.
è Deficiency or overproduction of hGH can lead to disease: dwarfism,
gigantism and acromegaly among other diseases.
o But is needed to grow.
Sequence and structure:
hGH is a protein (191 amino acids) = main isoform.
è But there are multiple versions synthesized in vivo (at least 5
different isoforms possible).
o An isoform is a protein variant differing in sequence (extra or less amino acids) or post-translation
modification. There can be differences in function between isoforms.
Previously: hGH was identified and they knew where it was, but not what it is. They started to purify it and found that
mice grew from it. Many therapeutics begin from extraction à purification à identify. But, hGH has a high degree of
species specificity. It needs to be human or from related primates.
How come those interactions are so specific?
ð Large surface areas contribute to binding à surface interacts with the receptor à specificity. There will be
entropic and enthalpy contributions.
o If you extract hGH from dead bodies, you will need 108 bodies to treat 1 patient for 3 years. This is not
sustainable + high levels of contamination.
o Also, purification of biologicals is never perfect à batches became contaminated with prions à can
cause diseases (ex. Prine disease, mad cow disease). Cadaveric extraction banned in 1985. So, a solution is
making it with biotechnology.
Central dogma:
If you want to produce a protein in vivo, you need its genetic information (DNA sequence).
è Bacteria: You have no RNA processing and very few proteins get modified. You
just need to find DNA sequence to make a protein.
è Eukaryote: here you have RNA processing and proteins get modified. Reverse
transcription of RNA -> DNA but important is reverse transcription of mRNA ->
cDNA.
hGH has 5 exons with at least 4 isoforms.
First research without biotechnology:
After finding a source of high level of protein, you will have a lot of RNA. Acromegalic tumors have a high level of growth
hormone synthesis.
1) You isolate mRNA and translate it in vitro (checked for size in gel and for aggregation in anti-serum).
2) The next step is cDNA synthesis from mRNA, and you will have a mixture of molecules present.
3) Then we will do cloning: S1 endonuclease + polymerase, ligation of HindIII adaptor and HindIII-digested plasmid.
è They synthetized hGH as a precursor protein and additional PTM’s but not ready for therapeutic use. Only
disulfide bonds are important but, cannot be made in bacteria.
3
, è There are a lot of next steps before it hit the market, after assembling DNA à from gene to biological à scaling it
up à purification on scale à formulation à validation and regulation. hGH first produced in 1979, clinical trials
from 1981 and therapeutic use from 1986.
Today:
There is more knowledge, databases and high-throughput methods for discovery. There is cheaper DNA synthesis and
better protein expression and purification tools. More robust molecular biology methods.
1) The gene sequence is already known
2) Search if it has been characterized or if it is in the genome
3) Sequence mRNA (if isoforms were not known) and obtain cDNA
4) Mass spectrometry/proteomics to determine if and which modifications are present
5) Process design: decide on host, expression tags, purification strategy and optimize sequence for the chosen host.
You don’t have to clone anymore and you will just order construct commercially. You express and optimize (ex.
changing a sequence). Each of these steps affect the final product.
Classic molecular biology:
Dependent on identifying suitable sites for restriction with endonucleases.
è Case-by-case answers/results
è Compromise generally required
è Expensive
è You need multiple enzymes
The pET system is generated by classic methods, has multiple cloning sites (MCS) and pre-cloned affinity tags
Synthetic biology (more than genetic engineering):
a) Traditional: you take a complex system and make this simpler until it breaks and that is your minimum system
minimize complexity to make system tractable (= top-down approach).
b) Synthetic: construct complex systems by assembling small components (= bottom-up approach).
SBOL = synthetic biology open language
Open standard for the representation of biological designs. It creates a common language to represent at an abstract
level, the information contained in the underlying DNA.
è Origin of replication = start of replication of DNA/RNA
è Endonuclease cut site = DNA sequence where an endonuclease (an enzyme that cuts DNA inside a strand) makes
a break in the DNA backbone
è Primer binding site = region of a DNA/RNA sequence where a primer binds to initiate DNA replication or
amplification
è Promotor = DNA sequence that serves as a binding site for RNA polymerase and other transcription factors to
initiate transcription.
è Operator = DNA sequence in a gene's regulatory region where repressor proteins bind to control gene expression.
è Ribosomal binding site (RBS) = sequence on mRNA where the ribosome binds to initiate translation
è Coding sequence = part of a gene or mRNA that contains the instructions for making a protein
è Polyadenylation site = sequence in eukaryotic mRNA that signals the addition of a poly(A) tail at the 3' end of the
transcript.
è Intron = non-coding sequence within a gene that is transcribed into RNA but removed during RNA splicing before
translation
è Translation stop site = point in an mRNA sequence where translation ends, signaling the ribosome to release the
newly synthesized protein
4
1
, H1: peptides, proteins and enzymes
1. Biologicals and biotechnology – drugs
How do they work?:
- Activate (agonist) or deactivate (antagonist) an enzyme: binding
directly to the active site or through allosteric binding
- Add or remove a metabolite (or analogue)
- Add or remove a protein
- Activate, change or deactivate a biological response (cytokines,
steroids, peptides, gases)
- Alter the biological system temporarily or permanently
Where do they work?:
Drugs work in on receptors on the surface of the cell.
è An oral drug goes through your stomach, intestine, blood and it starts spreading in circulating system.
Once it survives the enzymes from the mouth, the low pH in the stomach and the intestines it goes to
the liver, where it will continue to go kidney until it gets eliminated.
è For small molecules there is easy penetration, but biological molecules get bigger (peptides, antibodies,
viruses, bacteria, human cells … as drugs) and have it more difficult to pass the membrane.
Different types of drugs have a different time of working. It depends on how they come in the body and how
they are eliminated. Some molecules go through you (they will never go into your blood system) and you will
just excrete them again. Antibodies can stay 20-28 days in your body. This cannot be injected orally, but you
deliver them intravenously. For gene therapy, one injection in your lifetime suffices.
Differences between chemical vs biological drugs:
Biopharmaceutical = an active drug substance made by a living organism or from a chemical reaction involving biological
parts.
è By biotherapeutic you never get 100% of the same molecules, this is different from chemics. You rely on the cell
that makes the product for you. This is a long-complicated process with different steps. All these different steps
can affect your final product.
2
, 2. Human growth hormone
hGH (a.k.a somatotropin) is made in the pituitary gland (= hypophysis) and
released in the blood stream.
è It has a direct effect on many cell types and activates IGF-1
synthesis in the liver.
è Deficiency or overproduction of hGH can lead to disease: dwarfism,
gigantism and acromegaly among other diseases.
o But is needed to grow.
Sequence and structure:
hGH is a protein (191 amino acids) = main isoform.
è But there are multiple versions synthesized in vivo (at least 5
different isoforms possible).
o An isoform is a protein variant differing in sequence (extra or less amino acids) or post-translation
modification. There can be differences in function between isoforms.
Previously: hGH was identified and they knew where it was, but not what it is. They started to purify it and found that
mice grew from it. Many therapeutics begin from extraction à purification à identify. But, hGH has a high degree of
species specificity. It needs to be human or from related primates.
How come those interactions are so specific?
ð Large surface areas contribute to binding à surface interacts with the receptor à specificity. There will be
entropic and enthalpy contributions.
o If you extract hGH from dead bodies, you will need 108 bodies to treat 1 patient for 3 years. This is not
sustainable + high levels of contamination.
o Also, purification of biologicals is never perfect à batches became contaminated with prions à can
cause diseases (ex. Prine disease, mad cow disease). Cadaveric extraction banned in 1985. So, a solution is
making it with biotechnology.
Central dogma:
If you want to produce a protein in vivo, you need its genetic information (DNA sequence).
è Bacteria: You have no RNA processing and very few proteins get modified. You
just need to find DNA sequence to make a protein.
è Eukaryote: here you have RNA processing and proteins get modified. Reverse
transcription of RNA -> DNA but important is reverse transcription of mRNA ->
cDNA.
hGH has 5 exons with at least 4 isoforms.
First research without biotechnology:
After finding a source of high level of protein, you will have a lot of RNA. Acromegalic tumors have a high level of growth
hormone synthesis.
1) You isolate mRNA and translate it in vitro (checked for size in gel and for aggregation in anti-serum).
2) The next step is cDNA synthesis from mRNA, and you will have a mixture of molecules present.
3) Then we will do cloning: S1 endonuclease + polymerase, ligation of HindIII adaptor and HindIII-digested plasmid.
è They synthetized hGH as a precursor protein and additional PTM’s but not ready for therapeutic use. Only
disulfide bonds are important but, cannot be made in bacteria.
3
, è There are a lot of next steps before it hit the market, after assembling DNA à from gene to biological à scaling it
up à purification on scale à formulation à validation and regulation. hGH first produced in 1979, clinical trials
from 1981 and therapeutic use from 1986.
Today:
There is more knowledge, databases and high-throughput methods for discovery. There is cheaper DNA synthesis and
better protein expression and purification tools. More robust molecular biology methods.
1) The gene sequence is already known
2) Search if it has been characterized or if it is in the genome
3) Sequence mRNA (if isoforms were not known) and obtain cDNA
4) Mass spectrometry/proteomics to determine if and which modifications are present
5) Process design: decide on host, expression tags, purification strategy and optimize sequence for the chosen host.
You don’t have to clone anymore and you will just order construct commercially. You express and optimize (ex.
changing a sequence). Each of these steps affect the final product.
Classic molecular biology:
Dependent on identifying suitable sites for restriction with endonucleases.
è Case-by-case answers/results
è Compromise generally required
è Expensive
è You need multiple enzymes
The pET system is generated by classic methods, has multiple cloning sites (MCS) and pre-cloned affinity tags
Synthetic biology (more than genetic engineering):
a) Traditional: you take a complex system and make this simpler until it breaks and that is your minimum system
minimize complexity to make system tractable (= top-down approach).
b) Synthetic: construct complex systems by assembling small components (= bottom-up approach).
SBOL = synthetic biology open language
Open standard for the representation of biological designs. It creates a common language to represent at an abstract
level, the information contained in the underlying DNA.
è Origin of replication = start of replication of DNA/RNA
è Endonuclease cut site = DNA sequence where an endonuclease (an enzyme that cuts DNA inside a strand) makes
a break in the DNA backbone
è Primer binding site = region of a DNA/RNA sequence where a primer binds to initiate DNA replication or
amplification
è Promotor = DNA sequence that serves as a binding site for RNA polymerase and other transcription factors to
initiate transcription.
è Operator = DNA sequence in a gene's regulatory region where repressor proteins bind to control gene expression.
è Ribosomal binding site (RBS) = sequence on mRNA where the ribosome binds to initiate translation
è Coding sequence = part of a gene or mRNA that contains the instructions for making a protein
è Polyadenylation site = sequence in eukaryotic mRNA that signals the addition of a poly(A) tail at the 3' end of the
transcript.
è Intron = non-coding sequence within a gene that is transcribed into RNA but removed during RNA splicing before
translation
è Translation stop site = point in an mRNA sequence where translation ends, signaling the ribosome to release the
newly synthesized protein
4
