18. How can E. coli host cells be engineered so that complex proteins with a large...

18. How can E. coli host cells be engineered so that complex proteins with a large number of disulfide bonds are properly folded and therefore produced in an active form rather than as part of an inclusion body?

Expert Solution

Some of the available strategies for ensuring formation of properly folded disulfide-bonded proteins in bacteria such as E. coli are -

Periplasmic expression - The periplasm is an oxidizing compartment due to the presence of thioredoxin superfamily proteins and it hosts enzymes catalyzing disulfide bond formation and their isomerization, as well as specific chaperones and foldases. The fusion of recombinant proteins to suitable leader peptides allows for the translocation of unfolded precursors into the periplasm by either the Sec or the SRP system.
Modulation of the expression level - To avoid overcrowding in periplasm, protein secretion can be effectively modulated at the translational level by modifying the Shine-Dalgarno sequence.
By creating double mutant strains of thioredoxin/glutaredoxin reductase (trxB-, gor-) pathway - These strains are characterized by the presence of an oxidizing cytoplasm, compatible with the folding
of proteins that need disulfide bridges for stabilizing their structure.

gladiator answered 1 year ago

Compare and contrast how the initiation complex is assembled in bacteria (E. coli) and in eukaryotes (budding yeast cells).

Compare and contrast how the initiation complex is assembled in bacteria (E. coli) and in eukaryotes (budding yeast cells). How do the bacterial and budding yeast ribosomes determine which start codon to initiate translation? Include in your description a discussion of the cis-acting elements, trans-acting factors, and energy requirements required for building the initiation complex in each system. The best response for this question will NOT BE A LIST of the requirements. It will clearly COMPARE and CONTRAST this process...

Explain how synthesis of ribosomal proteins in E. coli is regulated at the level of translation.

Roughly how many soluble proteins are there in E. coli? Refer back to your Isolation and...

Roughly how many soluble proteins are there in E. coli? Refer back to your Isolation and Purification of GFP lab. Describe what proteins should be present in each of your gel samples (crude, flow through, wash and elution samples). What is the known size of GFP from the literature? How will you determine the size of your GFP by SDS-PAGE?

Consider a colony of E. coli cells in which the gene for the CRP protein is...

Consider a colony of E. coli cells in which the gene for the CRP protein is deleted. Describe the effect of that this deletion will have on the arabinose operon in terms of levels of Ara A protein (one of the proteins encoded by a gene in the ara operon) when the cells are grown in media containing arabinose but no glucose. Explain your reasoning.

PREPARATION OF COMPETENT CELLS and TRANSFORMATION: 2a. We will be preparing competent MM294 (E. coli) cells...

PREPARATION OF COMPETENT CELLS and TRANSFORMATION: 2a. We will be preparing competent MM294 (E. coli) cells using a chemical treatment of CaCl2. What are MM294 cells, why do we use them? Define “competent” cells and describe why/how CaCl2 makes cells “competent”. Why is “mid-log” phase important? Support your drawing with a cartoon of what cells “look like” before and after treatment. 2b. What does “heat-shock” do (in the transformation procedure)? 2c. What is LB? What does “LB” stand for and...

The E. coli trp operon encodes proteins involved in the biosynthesis of the amino acid tryptophan....

The E. coli trp operon encodes proteins involved in the biosynthesis of the amino acid tryptophan. Control of trp operon expression is mediated by two distinct regulatory mechanisms. Describe these two regulatory mechanisms in detail. Note Please give detail and comprehensive answer .

a) Describe how you could determine quantitate (determine how many) E. coli cells have been transformed?

a) Describe how you could determine quantitate (determine how many) E. coli cells have been transformed? b) Briefly outline your experiment. You will need a control, or standard, to analyze your experimental results, make sure to indicate this in your answer – explain what the control/standard is and how you use it.

Consider a colony of E. coli cells that have a mutation in the lac I gene,...

Consider a colony of E. coli cells that have a mutation in the lac I gene, resulting in a LacR protein that cannot bind to allolactose. Describe the effect that this mutation will have on the lac operon in terms of βgal protein levels when the cells are grown in media containing lactose but no glucose. Explain your reasoning.

Which is likely to occur in E. coli cells that are grown in milk (Hint: Milk...

Which is likely to occur in E. coli cells that are grown in milk (Hint: Milk contains lactose sugar)? The lac operon is shut off, and the cells will not produce lactose-utilizing enzymes. The lac repressor is inactivated, and the cells will produce lactose-utilizing enzymes The lac operon is turned on, but the bacteria will not produce lactose-utilizing enzymes. ---- wrong

Part A Escherichia coli (E. coli) can under optimal conditions double every 20.0 minutes. How long...

Part A Escherichia coli (E. coli) can under optimal conditions double every 20.0 minutes. How long would it take a single cell to saturate a 5.0 L culture assuming that optimal growth conditions prevaled and that no cells died? The maximum cell denisty in a saturated culture is 1010 cells mL-1. Enter your answer to the nearest tenth of an hour. Part B Consider the information in Part A and decide how long it would take for the 5.0 L...

Question