PREGUNTAS PROFESORA Flashcards
What is Inclusion bodies ? & Why do they complicate protein purification ?
- They are aggregates of misfolded or denatured proteins.
*The formation of IBs greatly reduces the yield of recombinant protein.
Factors influencing the formation of inclusion bodies : 7
- Expression level (up to 50% of total protein)
- Expression rate
- Temperature and thus indirectly the growth rate of the host
- Missing co-factors and chaperones (imbalance due to overexpression)
- Missing subunits.
- Missing disulfide bridges
- Composition of the media/carbon source (complex C sources are better because they lead to slower cell growth => fewer IBs!)
- What are the advantages of yeast as an expression system?
o Selection not via antibiotics, but via amino acids
o Modifications possible ( proteolytic processing , N-terminal blocking, glycosylation , farnesylation , phosphosylation , SS)
o Yeast-specific promoter and termination sequence
o Use of various tags (some improve solubility (GST) others improve folding and more specific and easier purification possible)
o High protein yield
o High level of secretion
o High cell density
- Intein -Chitin System expl
An elegant method for the affinity chromatographic purification of recombinant proteins. The target protein (TP) is fused N- or C- terminally with intein . Intein is a genetically modified peptide that has a chitin binding site on one side and can proteolytically cleave itself on the other side when induced with DTT and lowered by temperature.
The fusion protein of TP and intein can be isolated and concentrated on a column using chitin as the column material. By induction with DTT and lowering the temperature to 4°C, intein cleaves the TP proteolytically . The TP can now be eluted in the desired buffer , while intein itself remains on the column
- Intein -Chitin System Benefits?
Advantage:
* The TP is present in the eluate in native form, without fusion component -> Can be used for crystallography, NMR or therapeutic purposes.
* Apart from TP, the only thing present in the eluate is DTT, no salts or proteases! -> DTT can be easily removed, which facilitates the purification of the isolated protein.
* No use of proteases necessary to remove the fusion portion (these often cut non-specifically, making handling difficult).
- Where does disulfide bond formation occur in E. coli and yeast?
E. coli: In the periplasm
Yeast: in the endoplasmic reticulum
- Phosphorylation capacity in E.Coli ?
- Inefficient Post-Translational Modification
2.Phosphorylation of histidine residues is more prevalent in E. coli
3.E. coli is capable of phosphorylating Ser, Thr, and Tyr residues, but these modifications occur at much lower levels compared to eukaryotic cells.
These phosphorylations are involved in various regulatory processes but are less common than His phosphorylation. - E. coli contains homologs of Ser/Thr kinases found in eukaryotes.
These kinases facilitate Ser/Thr phosphorylation, although their roles are less extensive compared to eukaryotic counterparts.
B. subtilis
phosfolitaion
1.Similar to E. coli, B. subtilis primarily relies on His phosphorylation within its two-component systems for signal transduction.
The levels of Ser, Thr, and Tyr phosphorylation in B. subtilis are also lower than in eukaryotic cells.
ms for Ser, Thr, and Tyr phosphorylation but these are not as extensive or diverse as in eukaryotic cells.
In summary, while both E. coli and B. subtilis possess the capacity for protein phosphorylation, they do so with less efficiency and diversity compared to eukaryotic cells. His phosphorylation plays a dominant role in both bacteria, particularly within their two-component systems, whereas Ser, Thr, and Tyr phosphorylations are present but less prevalent.
- Purification of recombinant proteins from plants (list and describe points)
- Cell disruption -> relatively difficult due to the thick cell wall of the plant cells
- Cell lysate usually has a low protein concentration because it is extremely rich in carbohydrates (removal of carbohydrates by PEG precipitation).
- Another problem: Cell disruption releases many proteases and leads to rapid oxidation of the cell lysate (“ tanning ”) -> lysate turns black or brown. Therefore, use protease inhibitors (mostly have metalloproteases and not serine proteases like animals) or reducing agents such as DTT, ascorbic acid, beta mercaptoethanol etc.
- Purification of the target protein can then also be done by various chromatographic methods -> affinity chromatography, ion exchange chromatography, size exclusion , salting out/organic LM, reverse phase chromatography (hydrophobic WW.)
PHI31C Integrase in the Gateway System
PHI31C integrase is a key component of the Gateway cloning system used to facilitate the integration of interest genes into the genome of mammals by recognizing pseudo-att sites and integrating the expression cassette near housekeeping genes.
The PHI31C integrase recognizes specific sequences called attB (bacterial attachment site) on the vector and pseudo-attP (phage attachment site) sites in the mammalian genome.
The attB site is present on the vector containing your target gene, and the pseudo-attP site is naturally occurring in the mammalian genome.The PHI31C integrase catalyzes the recombination between the attB site on the vector and a pseudo-attP site in the genome.
This recombination leads to the integration of the vector’s DNA (including the target gene) into the mammalian genome at the pseudo-attP site.
- What are rCHO cells?
rCHO cells are genetically engineered Chinese hamster ovary cells that are optimized for high-efficiency production of recombinant proteins.
They are typically DHFR-deficient, so they cannot synthesize nucleotides de novo and thus cannot grow without an external source of these metabolites, which allows for a selective growth advantage for cells that carry the desired genetic modifications, making them a powerful tool in the production of therapeutic proteins.
rhco uses
rCHO cells are extensively used in the production of biopharmaceuticals, such as monoclonal antibodies, enzymes, and other therapeutic proteins. Their ability to grow in suspension cultures and their post-translational modification capabilities make them highly valuable in this field.
codon usage
codon usage plays a crucial role in the expression of heterologous proteins in prokaryotic hosts due to differences in codon preferences between species. Addressing this issue can be achieved either by optimizing the coding sequence of the gene to match the host’s preferred codons or by engineering the host to express tRNAs for rare codons, thereby enhancing the efficiency and yield of protein production.
(Commercially available strains of E. coli, such as those designed for high-level expression of eukaryotic proteins, often include genes for rare tRNAs.)
what is Glycosylation and why is it possible
in Saccharomyces cerevisiae
Glycosylation is a crucial post-translational modification where carbohydrate chains (glycans) are attached to proteins. In Saccharomyces cerevisiae , glycosylation is feasible because the yeast cells possess an endoplasmic reticulum (ER) and Golgi apparatus, as well as a functional secretory pathway
process of glycosylation in S. cerevisiae:
Glycosylation begins in the ER, where initial glycan structures are attached to nascent proteins.
These glycoproteins are then transported to the Golgi apparatus, where the glycans are further modified and matured.
Proteins destined for secretion follow a pathway that involves their transport from the ER through the Golgi to the cell surface or extracellular space.
