Recombinant Proteins Flashcards
1
Q
What are recombinant proteins?
A
Splicing of; 2 pieces of DNA stitched together (bringing together genetic material from multiple sources)
2
Q
Why use recombinant proteins as therapeutics?
A
- Protein perform complex and highly specific functions; nor easily mimicked by chemical drugs
- Humans proteins can be produced from non-human sources via recombinant techniques
- Relatively cost effective and limitless supply
- Facilitates protein engineering to improve stability, yield, ADME properties
- Reduced possibility of pathogen contamination (e.g. prions, HIV)
- Reduced possibility of immune rejection/allergic reactions due to species differences or contaminants
- Circumvents religious objections to animal products e.g. porcine insulin
- More rapid clinical development and regulatory approval compared to small molecules
3
Q
What are some clinical uses of recombinant proteins/biologicals?
A
- Replacing missing or defective protein e.g. in endocrine, metabolic disorders (insulin, growth hormone, clotting factors, metabolic enzymes)
- Enhance/augment existing pathways (interferons; MS, cytokines, erythropoietin; anaemia, HRT)
- Interfere with harmful molecules or proteins produced by the body (inhibitors of growth hormone receptors; acromegaly, TNFalpha blockers; psoriasis/RA, DNAse; cystic fibrosis, anti-clotting agents.
- Delivery of other proteins or drugs to a specific site/tissue (antibodies linked to cytocidal compounds selectively targeting cancer cells)
- Antiinfectives - antibiotics, antivirals (interferon α and γ, monoclonal Abs, vaccines)
- Eliminating other harmful foreign substances (anti-venoms, anti-toxins)
- Treat autoimmune diseases, inflammation, transplant rejection (anti-TNF-α; psoriasis, anti-rhesus immunoglobulin, interferon β; MS)
- Anti-cancer therapies (avastin, herceptin; breast cancer, rituximab; B-cell lymphoma)
- Promote tissues repair, regeneration (bone morphogenic proteins)
- Diagnostics/biomarkers of disease, infection, cancer, metabolic disorders (HIV, HPV, TB, PSA, GHRH, TSH, Glucagon)
4
Q
How has insulin progressed to recombinant human insulin?
A
1922 - Porcine insulin used to treat DM
> But animal derived insulins (cow, horse, pig) caused allergic reactions; low purity
1978 - Human insulin produced in E. coli
1982 - Eli Lilly markets ‘humulin’
5
Q
How are fast-acting insulins e.g. lispro/glulisine/aspart modified for their therapeutic use?
A
- They exist as monomeric forms; unable to form hexamers (when insulin binds zinc and clumps with other insulins)
- Alterations at C-terminus of B-chain (swap AAs)
- Lispro; Lys-Pro-Thr
- Glulisine; Pro-Glu-Thr
- OG Humulin; Pro-Lys-Thr
6
Q
How does the long-acting insulin glargine have such an effect?
A
- Modified A and B chains
- Form microcrystals/other aggregates
- Slow rate of monomer release
- Arg-Arg added onto OG Pro-Lys-Thr
7
Q
What is Gaucher’s disease and how is it treated?
A
- Rare congenital disorder of lipid metabolism
- Deficiency in β-glucocerebrosidase enzyme
- Lipids build up in macrophages
Symptoms: hepatomegaly, splenomegaly, bone lesions
Treatment: enzyme replacement therapy; infusion of the enzyme extracted from human placentas.
8
Q
How did recombinant proteins change how Gaucher’s disease was treated? What does this result in?
A
- Initially required 50,000 placentas to treat ONE patient for a year
- Production of recombinant form Cerezyme made treatment feasible
- Arginine 495 is changed to histidine
- This results in addition of mannose sugar
- Mannose recognised by cell surface receptors enhancing uptake in macrophages
9
Q
What is erythropoietin (EPO) and what can it be used to treat?
A
- Growth factor, stimulating erythrocyte (RBC) production
- Treats anaemia in CKD, and in IBD
10
Q
How can EPO be modified and what advantages does this bring?
A
- Change of two AAs introduces 2 additional N-glycosylation sites
- Half-life normally 5 hours; becomes 3-fold longer
- This meant fewer injections were required
11
Q
What can contribute to growth failures disorders that result in the under production/resistance to growth hormone (GH)?
How is it treated?
A
- CKD, growth hormone deficiency, Prader-Willi Syndrome, Turner syndrome
Treated with recombinant human growth hormone (GH) and insulin-like growth factor (IGF-1); daily injections.
12
Q
How does GHRH affect GH etc. to lead to growth?
A
- Hypothalamus releases GHRH (Growth Hormone Releasing Hormone)
- This stimulates the pituitary, which then releases GH
- GH acts at the liver causing the production of IGF-1 (insulin-like growth factor)
- This stimulates long bone growth, encouraging processes such as metabolism of fats and CHO = cell growth
13
Q
What is gigantism and how is it characterised?
A
- Symmetrical enlargement of the body; overgrowth of long bones, connective tissues, visceral organs
- Caused by excess GH/IGF-1 caused by benign pituitary tumours (adenomas)
14
Q
What is the difference between gigantism and acromegaly?
A
- Gigantism = excess GH/IGF-1 in early life
- Acromegaly = excess secretion of GH/IGF-1 after the body has stopped growing
15
Q
What characterises acromegaly and how can it be treated?
A
- Enlargement of bones in hands, feet, face
- Treated with somatostatin analogues, blocking GF production
16
Q
What areas of biomedical research use recombinant proteins?
A
- Protein structure/function studies
- Antibody production
- Drug discovery
- Recombinant DNA techniques
- Stem cell technology
- Gene therapy
- Bioimaging
- Disease biomarkers
- Translational research
17
Q
Define: Recombinant DNA.
A
- Fragments of DNA (or copy DNA) recombined to generate a synthetic molecule
- DNA from different species can be recombined
18
Q
Define: Host/vector systems
A
- DNA of interest inserted into plasmid or viral vector
- Vector introduced into host cell/organism
- Vector contains a gene promoter functional in host organism
- Expression of recombinant proteins for medical use/reseach
19
Q
Define: Recombinant proteins
A
- Proteins generated from recombinant DNA vectors
20
Q
Define: Transgene
A
- Recombinant DNA that is introduced into the genome of another organism
21
Q
Define: Genetic engineering
A
- Alteration of DNA sequences to change gene function or expression
- In vitro/in vivo methods to combine DNA sequences from different organisms
22
Q
What is a GMO?
A
Genetically modified organism
23
Q
What is DNA and how is it transcribed to RNA?
A
- Pair of antiparallel chains
- ATGC
- Adenine
- Thymine
- Guanine
- Cytosine
24
Q
What is transcription?
A
The process of making a copy of the anti-sense DNA strand into a sense RNA strand
25
How are proteins made from RNA?
- Via tRNA in ribosomes
| - Translation of RNA to protein
26
What are prokaryotes and what is their gene structure?
- Bacteria
DNA:
- PROMOTER; transcription start site for RNA polymerase to start synthesising RNA at
- AUG start codon for translation in ribosomes
- Open Reading Frame (ORF); (the Cistron/gene)
- Stop codon for translation in ribosomes
- TERMINATOR; transcription stop site for RNA polymerase
mRNA:
- Random regulatory region not translated e.g. attracting ribosomes (bit between Promoter and ORF from DNA?)
- ORF (Cistron)
- Random regulatory region
Protein:
- Linear sequence
- Displayed from N to C terminus
27
Describe mammalian gene structure.
dsDNA (double-stranded):
- Transcription Start Site (TSS)
- Introns (non-coding parts)
- Exons (protein coding parts)
- Transcription Termination
```
pre mRNA (primary transcript):
- Whole gene transcribed inc. non-coding introns
```
mRNA (processed; capped and spliced):
- pre mRNA is processed down to much smaller size
- Introns removed by spliceosome
- 5' end capped w/7-methyl Guanylate cap
- 3' end cleaved and PolyA tail (adenine nucleotides) added (increases stability/prevents degradation)
- Untranslated regions at either end
- CDS/coding sequence in middle
- Primary transcript now processed to mRNA
28
What techniques can genetic engineers employ?
- Restriction/modification enzymes
- DNA ligase (stitching DNA together)
- Reverse transcriptase (make DNA copy from RNA)
- PCR
- DNA sequencing
- Plasmid/viral vectors
- Controlled expression systems
29
What are restriction enzymes?
- Bacterial endonucleases
- Restrict growth of viruses infecting bacteria
- Cleave/cut specific DNA sequences
- Often recognise palindromic sequences (reads same backwards/flipped)
- Evolved as a defence against bacteriophage infection (killing viral infection in bacteria)
- Co-evolve with modification system to protect self (self-immunity)
30
What is the difference between endonucleases and exonucleases?
Endonucleases: cleaves DNA in middle etc from recognising sequence
Exonucleases: doesn't look for specific sequence; only the end of the molecule and chops it uo
31
How do restriction enzymes e.g. EcoR1 modify itself to prevent self-cleave?
- Modification entails adding methyl group to relevant palindromic sequence that is potentially shared with foreign/invading virus
- Methylation protects own genome from endonucleases
32
How do restriction enzymes e.g. EcoR1 cleave DNA and what can happen to the products?
- Non-uniformly, like sideways Z
- Generates cleavage products with a 5' overhang
- Single-stranded AATT followed by dsDNA
- Products complementary to each other; can be annealed by DNA ligase in the presence of ATP
33
How do blunt ends differ from cohesive ends (from restriction enzyme cleavage)?
- Blunt ends; cleaving both chains at same position
- Thus any 2 blunt ends can be ligated together with DNA ligase (+ ATP); they are not sequence specific
- However cohesive ends require sequence compatibility (if they have complementary base pairs) e.g. EcoR1 will only ligate with another EcoR1
34
What host-vector systems are available for recombinant proteins?
- Bacterial cells (E.coli, Corynebacterium spp., Bacillus spp.)
- Yeast cells (Pichia, Saccharomyces cerevisiae, S. pombe)
- Cultured mammalian cells
- Transgenic plants and animals (pharming)
- In vitro systems
> DNA of interest is introduced into cell/organism so that RNA can be generated to make the protein
35
What is a cloning vector?
A piece of DNA that is selectable that can carry your gene of interest, and make sure it's expressed in the host cell system.
36
What are examples of cloning vectors?
- Bacterial plasmid DNA
- Bacterial viruses (bacteriophage DNA)
- Yeast plasmids
- Animal viruses (retrovirus, lentivirus, adenovirus)
> Process referred to as 'DNA Cloning'; generation of many identical copies of a gene or piece of DNA
37
What barriers are present in making recombinant proteins from inserting human DNA into bacteria?
- Human genes contain introns and exons
- Bacteria cannot process introns (can't splice them out)
- Bacterial RNA polymerase cannot recognise mammalian promoter sequences (that drive expression of genes)
38
What solutions are available to overcome the issues of making recombinant proteins from human DNA in bacteria?
- Remove intron sequences
- Or use cDNA (copy DNA instead of cloning genomic DNA); a copy of the processed mRNA
- Insert into a vector behind a bacterial promoter
39
What does cDNA/transcript cloning entail?
- processed mRNA transcribed from dsDNA (full human gene), undergone splicing to remove introns etc
- From mRNA transcript a DNA copy (cDNA) is made
- cDNA ligated into vector
> Only transcript is taken instead of whole gene
> 'Transcript cloning' more accurate than 'gene cloning
40
What enzyme is used to make cDNA from mRNA?
- Via reverse transcriptase enzyme
- Enzyme found in animal RNA viruses (retroviruses; unusual viruses that have their genome as RNA instead of DNA
- When they affect a mammalian cell they have to convert their RNA genome back into DNA so it can be expressed by human host
- Thus reverse transcriptase enzyme taken from animal viral cells and used in vivo to generate DNA from RNA
41
How is cDNA made from processed mRNA?
- mRNA tales all have PolyA tales
- Add in short sequence poly dT primer (or other specific primer), hybridises and anneals to PolyA tail; allows reverse transcriptase to synthesise DNA from mRNA template
- Reverse transcriptase makes a copy of the mRNA (cDNA)
- Want to rid of mRNA now; degrade it by adding NaOH to leave single-stranded DNA
- Add random primers (short 6 base pair oligonucleotides) binding randomly to ssDNA
- Allows DNA polymerase to make a copy of cDNA, resulting in dscDNA (double-stranded)
42
What is the basis of PCR?
- Thermostable DNA polymerase (thermus aquaticus; likes heat and water, happy at 70-80 degrees etc)
- Amplification (multiple copies) of a specific DNA sequence
- Temperature cycling
- PCR makes multiple copies exponentially, can use the enzyme again and again
43
What is a primer?
- Every DNA polymerase needs a primer
- It is a short DNA (oligonucleotide) sequence usually 15-30 base pairs long
- Complementary to the strand you're copying
- Primer annealing/hybridisation (base pairing) to its complementary sequence
44
What are the steps of PCR?
- Start off with PCR Reaction Mix: Template, Primers, dNTPs (nucleotide triphosphates containing deoxyribose), Taq Polymerase Enzyme
- Temperature denaturation at 94 degrees; dsDNA denatures
- Cooling down to 37 degrees quickly; double strands don't come back together because too long
- But primers will anneal to complementary system to each chain
- Bring temperature back up to 72 degrees; optimal temperature for Taq Pol Enzyme, synthesises DNA
- Leaves you with 2 strands of DNA instead of the starting 1
- 30 second process
- Second cycle start with 2 strands of dsDNA; denature/anneal/synthesise and end up with double number of copies
- Repeat for 35 cycles (thermal cycling; 94/37/72 degrees)
45
What other uses do PCR Primers have?
- Can be used to insert Restriction Enzyme sites e.g. annealing/hybridisation of EcoR1 into a sequence, engineering changes
- Can be used to mutagenesis/introduce sequence mutation, e.g. to swap AAs in insulin lispro; mismatch primer (but w/enough annealing to stick) generating a sequence change
46
What is bacterial plasmid DNA/does it get passed on to daughter cells?
- Extrachromosomal DNA
- Very small 'mini-chromosomes'; 1kb to several Mb in size (kilo-base)
- Autonomously replicating bacteria
- Circular double stranded DNA molecules
- Behave independently from OG genome
- Daughter cells of replicating DNA get full genome each and plasmid DNA too
47
What are the typical genes that plasmids carry?
- Antibiotic resistance genes
- Virulence factors (bad shit e.g. endotoxin)
- Metabolic enzymes
- Plasmid transfer
48
What is the significance of horizontal gene transfer (F plasmid)?
- Bacteria can share plasmids
- Even cross-species
- Leads to antibiotic resistance etc
49
How are plasmid vectors modified in genetic engineering?
- Adding a Promoter region (for bacteria, yeast, mammals)
- Adding restriction enzyme sites; allows insertion of different bits of DNA
- Antibiotic resistance e.g. Amp, Tep, Kan for resistance
50
What does DNA cloning entail?
- Insertion of DNA fragment into plasmid DNA
- DNA fragment (usually from PCR) is digested and treated with restriction enzymes
- Plasmid DNA also cut with the same restriction enzyme
- Then ligation can occur via DNA ligase (complementary as cut with same restriction enzyme)
- Plasmid DNA expresses gene behind bacterial promoter
51
What does blunt end ligation require?
- All PCRs have blunt ends; thus restriction enzymes not required for DNA cloning
- But will consequently require plasmid DNA to be cut with blunt-end generating restriction enzyme
- PCR fragment can be inserted in either rotations, but the wrong rotation won't be read as the promoter will be on the wrong side
- Thus directionality is important
52
How does cohesive/sticky end ligation differ from blunt end ligation?
- Instead of 50/50 yield with one half having the promoter at the wrong end, 100% yield can be achieved
- Via cutting ends of PCR fragment/plasmid DNA with a different restriction enzyme each e.g. EcoR1 and BamH1
- Forces direction to give single desirable product, ensuring correct orientation
- Two different restriction enzymes required as using just one means both ends are complementary = wrong rotation
53
What does Sanger Sequencing/the Chain Termination method entail?
What is used now?
- Spike mixture with dideoxy NTPs which are modified NTPs which can no longer form chain elongation
- Results in chain termination
- 100:1 ratio
- Denature and separate daughter strands by electrophoresis
- Runs through acrylamide gel according to molecular weight
- Fluorescent automated system now in place
54
How do we get the treated plasmid vector into the host?
Bacterial transformation:
- Treatment with CaCl/MgCl2 forms holes in the bacterial cell wall
- Heating to 42 degrees makes plasmid DNA enter bacterial cell
- Cells recover and then express the genes carried by the plasmid
- Antibiotic resistance gene included to find which cells have taken up the plasmid
- All cells that didn't take up the plasmid die, and the one's that did (desirable) grow because resistant
55
How do bacterial expression vectors differ from mammalian expression vectors?
Both can:
- employ different selective markers (e.g. antibiotic resistance)
- Have multiple different restriction enzyme sites e.g. lacZ' in bacterial; allows for insertion of whatever DNA code etc with ease
- Can be tagged with epitope/affinity tags to monitor expression
- Have different inducible promoter systems; thus gene might only be expressed in the mammalian cell and remains dormant in bacterial due to mammalian cell promoter
56
How can LacZ be used to our advantage to increase efficiency/yield of recombinant DNA production?
Detection of cloned inserted DNA:
- LacZ comes from E.coli, codes for β-galactosidase activity
- LacZ engineered to generate multiple cloning sites without compromising protein function
- DNA fragment treated with EcoR1 as well as host plasmid
- Ligation; plasmid can ligate back together without taking in DNA fragment = LacZ gene not compromised and xCAL gel turns white
- But if DNA fragment taken up; disrupts LacZ gene reading, xCAL colonies remain white instead
- Indication that white colonies have taken up the DNA fragment/insert; ones of interest
57
What are the reasons behind controlling plasmid gene expression in a host cell?
- Maximise yield and solubility
| - Minimise toxicity to host cell; keeping expression silent if toxic
58
What are inducible expression vectors and how do they work?
- Plasmid inert without the addition of an inducer; is repressed at promoter
- Upon activation with a small molecule, the repressor is removed and the gene is expressed e.g in presence of TPTG/doxicycline only
- Controls where/when the recombinant protein is made
59
How do we identify and detect recombinant proteins in vitro? What other role does it serve?
Immunopurification:
- Western Blotting: use antibody specific to the protein to detect it
- Or add epitope tag which fuses to protein of interest where a specific antibody immobilised on a matrix detects the tag instead
- Purifies 'soup' of bacterial enzymes and shit from E.coli etc too with highly specific antibody capturing protein of interest
60
What are the steps of Western Blotting?
1. ) Electrophoresis via SDS PAGE
2. ) Proteins transferred to nitrocellulose sheet in blotting tank; covalently bond to sheet holding OG positions of electrophoresis by MW
3. ) Add/incubate with first antibody that recognises the desired protein, washing excess
4. ) Incubate with enzyme-linked second antibody which has fluorescent tag or something that recognises first antibody
5. ) React with substrate for second antibody; showing fluoro tags etc.
61
When are epitope tags added in the instances of a protein not having a corresponding antibody for detection?
Epitope tag sequences added in the primer by PCR
62
What are the common problems/challenges with generating recombinant proteins?
- Differences in codon usage reduces translation efficiency (bacteria & mammals have different preference for tRNA codons; might have to engineer human sequences to more resemble bacteria e.g. CGA to CGG)
- Yield limited by toxicity of product to the host cell (thus control expression)
- Incorrect folding - poor solubility/aggregation
- Proteolytic degradation
- Absence of disulfide bonds
- Contamination; bacterial proteins, other molecules copurify
- Modified proteins may be immunogenic (evoking immune response)
63
Choosing the right/vector expression system; what is insulin/HGH grown in?
Insulin in bacterial cells,
| HGH in mammalian cells
64
What are the advantages of bacterial expression systems?
- Rapid growth in relatively inexpensive culture media
- Scale up easily in large fermenter
- Well defined genetic tractability (know how to manipulate them)
- Absence of contamination by animal/human viruses; very safe (for clinical use)
65
What are the disadvantages of bacterial expression systems?
- May lack posttranslational modifications
| - Correct folding/tertiary structure may be difficult to acieve
66
What are the steps used in generating a recombinant protein?
- Human tissue
- Isolate mRNA transcript of interest (not the gene)
- Generate cDNA using reverse transcriptase
- PCR amplification (and modify w/restriction enzyme sites)
- Subclone in Expression vector
- Transform E.coli or other host cell (getting plasmid in)
- Induce protein expression/production
- Optimise harvesting/protein purification
- Scale up Fermentation
- QA (quality assurance) and validation
- Preclinical trials
67
What is the pathway from idea of drug to patient?
- Preliminary research; investigate idea or hypothesis, if successful formal project established. May also buy in idea or technology from universities/research institutes/SMEs
- Research; develop production method; validation in closest animal models, experimenting with mutations
- Industrial manufacture; optimisation of production under increasing quality requirements, development of analytical methods to assess quality/activity
- Preclinical trials; toxicity studies in all organ systems (animal models)
- Clinical trials; phase 1, 2, 3
- Product registration; 12-18 months
- Launch
- Phase 4 trials; surveillance of long term side effects, treatment experience
> 7 - 10 years
68
What are the potential risks with recombinant protein therapeutics?
- Unintended (off target) effects in the body e.g. cancer promoting, toxic effects
- Allergenicity, toxicity
- Extensive preclinical testing of products in animals is mandatory before clinical trials in patients
- But problems may only arise during clinical use
69
What are the risks from industrial scale recombinant protein production?
- Large scale production of GMOs; could result in accidental environmental release? Damage to environment, animals, man?
- Inadvertent transfer of genes to other species e.g. pathogens
- Mutation of the GMO into a harmful organism
70
How are risks reduced in industrial scale recombinant protein production?
- Bacteria and yeast GMOs are engineered to be inviable in the environment (can't grow; essential genes removed so unable to survive outside the lab)
- E.coli strains used are unable to persist in human gut
- Engineered to have reduced capability for DNA transfer or mutation
- Product may be produced in an inert or inactive form
- High standards of containment, inactivation of waste products
71
What is the HSE?
- Health and Safety Executive; oversees all GMO generation/use with strict legislation for industrial production/research
- Risk assessed and subject to appropriate containment
- Level 1 = negligible/no risk
- Level 4 = high risk
