Term
| pancreatic hormone insulin has been used to treat which condition since 1922? |
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Definition
| diabetes type I becase of its effect in lowering blood glucose levels |
|
|
Term
| during the first decade of insulin therapy which two types if insuline were used? |
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Definition
|
|
Term
| name the 2 major advantages of human insulin? |
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Definition
it can be produced in limitless quantities cheaply
it is identical to native insulin so cannot provoke an immune response |
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Term
| insulin consists of 2 polypeptide chains (A and B). what bonds are these chains connected by? |
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Definition
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Term
| the 2 polypeptides of insulin are produced seperatley in E.coli a B-galactosidase fusion proteins. why is this fusion protein used? |
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Definition
| to stabilise the polypeptides as they are unstable on their own |
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Term
| after the fusion proteins are purified, the B-gal is cleaved off with which molecule? |
|
Definition
cyanogen bromide.
the A and B peptide chains are then purified and mixed. folding and oxidation of cysteine residues form disulphide bonds |
|
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Term
| give four reasons why foreign preoteins are often expressed as fusion proteins in E.coli? |
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Definition
- allows higher expression levels (rates of translation)
- > stability of expressed protein particularly if they are small
- may allow simple purification of recombinant protein
- may allow secretion of expressed protein
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Term
| in relation to vectors for expression of fusion proteins, why is there a protease cleavage site in between the fusion tag on the N-terminal and the multiple cloning site? |
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Definition
| the protease cleavage site allows the fusion tag to be removed later |
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Term
| vectors for expressing fusion proteins come in sets of three. why is this? |
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Definition
| so one of them will create the correct reading frame for the insert. |
|
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Term
| name 3 fusion tags that facilitate protein purification |
|
Definition
- polyhistidine (his-tag)
- maltose binding protein (MBP)
- Glutathione-S-transferase
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Term
| a his-tag consists of 6-10 histamines. name the ligand and elution involved in protein purification involving a his-tag |
|
Definition
ligand : nickel
elution: imidazole |
|
|
Term
| name the ligand and elution in protein purification involving a Maltose Binding Protein |
|
Definition
ligand: Amylose
Elution: Maltose
MBP size: 40kDa |
|
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Term
| whihc ligand does Glutathione-S-transferase bind to and what is the elution? |
|
Definition
ligand: Glutathione
Elution: reducing agent
size of Glutathione-S-transferase: 25kDa |
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Term
| what aparatus would you use to purify a protein with a MBP? |
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Definition
| an amylose-agarose affinity column |
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|
Term
which of the following proteases used for cleaving fusion proteins do not result in a N-terminal extension?
- Tev
- PreScission
- Thrombin
- Enterokinase
- Factor Xa
|
|
Definition
enterokinase and factor Xa
thrombin and preScission extend the N-terminal by 2 sequences
and Tev can vary the extension between 1 or 2 sequences. |
|
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Term
| sometimes expression of large amounts of proteins in E coli results in protein precipitating out. which process does this make more difficult? |
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Definition
| purification is made difficult |
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Term
| which peptide can be used to export a protein to the periplasm? |
|
Definition
a signal peptide.
the signal peptide is removed by host proteases as it is exported. this methodology is particulary applicable to proteins that are normally targeted towards the secretory pathway |
|
|
Term
| a signal peptide has 3 domains: N H and C. what are theses domains? |
|
Definition
- N domain - 2-15 positively charged amino acids
- H-domain - hydrophobic amino acids
- C-domain - here you have the signal peptidase recognition site
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Term
| name 2 therapeutic proteins the signal peptide OmpA is associated with |
|
Definition
Growth hormone
tPA - tissue Plasminogen Activator
the singal peptide is removed as the protein is exported |
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|
Term
| name 3 ways protein engineering can improve stability of a recombinant protein |
|
Definition
- > thermostability
- > stability at lower/higher pH
- resistance to oxidative inactivation thus longer shelf life
|
|
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Term
| name 3 ways protein engineering can improve kinetics in a recombinant protein |
|
Definition
- > affinity for substrate
- > speed of reaction
- altered substrate specificity
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Term
| in order to make a 'rational design' what must we know? |
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Definition
| detailed knowledge of protein structure and reactions |
|
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Term
| name two ways we can make defined changes in the coding sequence |
|
Definition
site-directed mutagenesis
artificial gene synthesis |
|
|
Term
| what was the name of the first synthetic organism to be created in may 2010? |
|
Definition
Mycoplasma laboratorium
this involved synthesis of a chromosome of over 1,000,000 bp! OMG |
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|
Term
| what is tPA -tissue plasminogen used for? |
|
Definition
| consisting of serine proteases, this is used to lyse blood clots (thrombolysis) particulary in heart attacks. it is cleared rapidly from bloodstream (50% in 5min) so has to be administered continuously |
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|
Term
| wht affect has recombinant engineered tPA had? |
|
Definition
| recombinant tPA engineered has increased its in vivo stability |
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|
Term
| which alkaline protease is extracted from Bacillus Subtilis? |
|
Definition
| Subtilisin - this is a stain remover in 95% of bio washing powders. |
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|
Term
| Subtilisin is expressed from recombinant DNA and has been engineered by which method to give which desired benefits? |
|
Definition
engineered by site-specific mutagenesis to be:
- stable to 70C
- resistant to non-ionic detergents
- resistant to oxidation (longer shelf life)
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|
Term
true or false
microbial biotechnology cannot be used for production of whole cells? |
|
Definition
False - it can eg.
Lactobacillus species are starter cultures for yoghurt/cheese manufacture
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|
Term
| Bacillus thuringenis is used to make what? |
|
Definition
|
|
Term
| single cell protein (SCP) can be used to culture which protein-rich meat substitute? |
|
Definition
|
|
Term
| from which microbial organism can you generate artificial snow from? |
|
Definition
Pseudomonas Syringae
this bacterium colonieses plant surfaces. some strains carry a membrane protein 'ice nucleation protein' (INP) that acts as a template for the formation of ice crystals |
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Term
| P syringae can initiate crystalisation at temperatures as high as -3C. Pure water can be cooled to which temperature without freezing? |
|
Definition
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|
Term
| when P syringae colonise the leaf surface it stimulates freezing. what does this do to the leaf? |
|
Definition
| it damages the leaf epithelium and makes the nutrients in the underlying plant available to the bacteria |
|
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Term
| what is Frostban™ and what does it contain? |
|
Definition
| it was one of the earliest GMOs (genetically modified organism). It contains an ice nucleation protein defective mutant for prevention of crop frost which displaces the wild-type strains |
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|
Term
| which polysacharide extracted from Acinetobacter is used in cleaning oil spills and enhances oil recovery? |
|
Definition
|
|
Term
| name the polysacharide extracted from Pseudomonas species that is used in the solidification of food products |
|
Definition
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|
Term
| which polysacharide is used as a food additive for stabilising liquid suspensions eg. ice cream? |
|
Definition
| Xantham gun from Xanthomonas species |
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|
Term
| name some compounds that are normally produced in limited quantaties. these are also known as metabolic intermediates |
|
Definition
amino acids, vitamins.
eg. L-glutamate for monosodium glutamate - flavour enhancer in chinese foods
L-phenylalanin+L-aspartate for artificial sweetner aspartame
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|
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Term
| name some compounds that are produced in excess as metabolic end products |
|
Definition
ethanol, organic acid
eg. citric acid in food and beverages, pharmaceuticals
Ascorbic acid (vitamin C)
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|
Term
| other microbial biotechnology products include antibiotics, pigments and toxins. what are these also known as? |
|
Definition
|
|
Term
|
Definition
using microorganisms to convert one organic compund to another
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|
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Term
in the synthesis of Prednisone (aniti-inflammatory drug used to treat autoimmune diseases such as arthritis) from Diosgenin what are the two ways it can be done? |
|
Definition
Chemical synthesis 37 steps $200/g
microbial bioconversion 11 steps $6/g |
|
|
Term
| where is diosgenin extracted from? |
|
Definition
| mexican yams. you can synthesise therapeutic steroids from diosgenin |
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|
Term
| the process of cleaning up contaminated sites such as sewage treatment, chemical degradation in soils and oil spills using microorganisms is known as? |
|
Definition
|
|
Term
| how is bioremediation of oil spills carried out? |
|
Definition
| fertiliser is sprayed to stimulate growth of indigenous bacteria that can breakdown the oil. this is the fastest and cheapest way to clean up beaches |
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|
Term
| name three therapeutic recombinant proteins |
|
Definition
Insulin
Erythropoietin
tPA (tissue plasminogen activator) |
|
|
Term
| name 3 reasons for expressing recombinant proteins as fusion proteins |
|
Definition
greater stability
increased expression
simpler purification |
|
|
Term
|
Definition
Polyhistidine (His-Tag)
Maltose Binding Protein (MBP)
Glutathione-S-transferase |
|
|
Term
name 3 ligands used in purifying fusion proteins
|
|
Definition
Nickel - His-Tag
Amylose -MBP
Glutathione - Glutathione-S-transferase |
|
|
Term
| what protein sequence is used to direct a protein sequence? |
|
Definition
|
|
Term
| what enzyme is used in biological washing powder? |
|
Definition
|
|
Term
| which species is used to culture microbial insecticide? |
|
Definition
| bacillus thuringensis (Bt) |
|
|
Term
| name the ice-necleating bacterium |
|
Definition
|
|
Term
| name the precursor for steroid production by microbial bioconversion |
|
Definition
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