Lecture 2 Flashcards
Production of proteins
Steps in protein production:
To obtain the proteins, we must first separate them from the rest of the cellular components and, once the protein pool is obtained, we will use chromatographic techniques to isolate the protein of interest.
Chromatographic techniques for protein isolation:
-Absorption: affinity separation of the protein to the matrix by hydrogen bonds and/or Van der Waals forces –> Gradient elution by increasing phosphate concentration.
-Affinity: specific interaction with the ligand covalently bound to the matrix –> Elution by cofactors, substrates, changes in pH or salt concentration.
-Metal chelation: by matrices containing bound metal ions for which the protein has an affinity –> Elution with ligand solutions that bind to the metal and displace the protein.
-Gel filtration: separation by molecular size (bulky proteins elute first) with a stationary phase composed of porous polymers.
-Ion exchange: separation according to charge (isoelectric point) by electrostatic forces dependent on the pH of the solution –> Cellulose matrix replaced by charged groups (cations or anions) or gradient elution by increasing the phosphate concentration.
-Hydrophobicity: by interaction with matrices containing hydrophobic functional groups –> Elution by altering ionic strength or pH change.
-High performance liquid chromatography (HPLC): at high pressure using columns with silica matrices.
what does dialysis and electrodialysis allow?
Removing inorganic ions and/or small solutes from the preparation by osmosis and/or use of membranes coated with ion-exchange resins
How many columns are used for the purification of proteins?
Almost no protein can be separated using a single chromatography column, but several columns are used, detecting the fraction in which the desired protein is found, and using it in another column, until the purified protein is finally obtained. Once obtained, the purity and specific activity must be verified.
What degree of purity do we need for the protein?
It depends on the application of the protein, being low in industrial applications (85-90%) but high in science, food or pharmaceuticals (100%). After purification, the ideal storage temperature should be checked, so that no loss of activity occurs, as well as the effect of additives.
Production of recombinant proteins
The production of recombinant proteins in heterologous hosts requires genetic manipulation, although the purification is the same as if the host were native, with the advantage that the yield is higher, as more protein is produced, we must collect all the fractions to check the procedure.
native expression (+) Vs. heterologous expression (-)
+ Correct functional structure
+ Constitutive expression
+ Limited production
+ Post-translational modifications
+ Native protein
____________________
- Undefined functional structure
- Constitutive or inducible expression
- Overexpression
- Unmodified in prokaryotes
- Inclusion bodies
what are affinity tags used for?
Affinity tags are a series of additional amino acids that are added to the end of the recombinant protein to facilitate purification processes by allowing, for example, the use of a single purification column. They can be added to both ends of the protein, but must be checked to ensure that they do not alter the activity of the protein. In many cases, they have associated linkers that provide cleavage sites recognised by specific proteases, allowing the affinity tail to be removed.
how can protein production be improved?
To improve production, host modifications such as improving transport systems, increasing the efficiency of production systems, modifying the chaperone content (facilitates protein folding) or eliminating proteases can be used to improve production.
is there a problem in expressing eukaryotic proteins?
When expressing eukaryotic proteins we have a problem, as the protein is determined by its mRNA (without introns) which requires the use of reverse transcriptase to obtain cDNA from the modified mRNA. There are also differences in size due to post-translational modifications.
types of restriction enzymes
Class I: cleave 100-1000 pb away from the recognition site and currently are or limited use
Class II: cleave very near a specific recognition sequence and are used to cleave double standed DNA at specific sites; they consist of one protein which cleaves the DNA and the second one methylates the DNA
Class III: cleave 25/27 pb from their recognition site and are gaining popularity in some cloning strategies; they carries out both funtions modifications and restriction (dependentofATP)
Where should these restriction enzymes be produced?
Several restriction enzymes can be produced by the same micro-organism, which makes purification processes difficult (→ must be 100%). Heterologous production of these enzymes in micro-organisms such as Escherichia coli is therefore useful; however, the latter must be protected from the restriction enzyme itself, e.g. by adding a suitable methyltransferase to the host.
Among the enzymes used in the food industry, I would like to talk about transglutaminase.
Transglutaminase is an enzyme that allows proteins to fuse by joining the CO - NH2 of glutamine with the NH2 group of lysine (with ammonium release) generating interconnected chains that provide greater water retention or elasticity, as well as preventing the formation of foams. It is obtained from Streptomyces as it has better temperature or pH ranges. It is generally used in food products, especially meat products, but can be useful to generate structures to grow human cells to repair damaged tissues.
enzymes for biofuel production
There are plant enzymes for the degradation of plant fibres to obtain sugars that can be converted into fuels by fermentation (1st generation biofuel). Later, microorganisms were discovered that produced these enzymes naturally, so they carry out the production of sugars and their transformation (2nd generation biofuel).
