Question

2. You are trying to design a protein that will be expressed in Escherichia coli and secreted outside of the cell for purification and use as a pharmaceutical. E. coli is a gram-negative cell and the protein folds after it has exited the cell. Which secretion system would work best for this project? Support your answer with evidence based on the properties of E. coli, the protein, and the secretion system. (20 pts)

2. You are trying to design a protein that will be expressed in Escherichia coli and secreted outside of the cell for purification and use as a pharmaceutical. E. coli is a gram-negative cell and the protein folds after it has exited the cell. Which secretion system would work best for this project? Support your answer with evidence based on the properties of E. coli, the protein, and the secretion system. (20 pts)

Answer 1

The requirements for the production of the protein are that it should be secreted outside the cell , it should be well folded (structural complexity and stability) and it carries pharmaceutical importance. According to me, to serve all the above 3 needs , the use of Type II secretion system (T2SSs) employing the leaky outer membrane mechanism will be most suited. The rationale behind this suggestion is as follows :

1. Type II leaky outer membrane (herewith referred as Type II Leaky OM) secretion system is a two-step secretion system which is constitutively expressed by E coli. Thus there is no need of provision of any special media or manipulation of environmental conditions for the production of desired protein.

2. This mechanism doesn't require an extracellular host i.e. the proteins are secreted from the cytoplasm to the extracellular space.

3. The proteins secreted through this mechanism do not need any secretion tag cleavage site thus making the process of secretion more efficient.

4. The proteins undergoing this secretion pathway have access to folding chaperones which ensures that the proteins are well folded and have structural complexity along with stability.

5. Engineering a leaky outer membrane allows for efficient protein production without significantly increasing the complexity process. Wurm et al. has formulated the optimal protocol of post culture incubation in 350 mM in Tris for several hours followed by a mild heat shock at 38 °C. This process released 30% of periplasmic proteins with < 5% cell lysis1.

6. Type II Leaky OM approach was demonstrated to secrete a high quantity of about 680 mg/L of Human Parathyroid hormone for therapeutic use2.

7. Type II Leaky OM mechanism has a broad specificity and is capable of secreting a diverse array of substrates.

8. This mechanism is well characterised i.e. well researched with comparatively simple structure.

9. Since proteins directly leak through the Outer Membrane, this mechanism produces high titers of substrates.

10. This system of secretion is less complex.

References :

1. Wurm DJ, Slouka C, Bosilj T, Herwig C, Spadiut O. How to trigger periplasmic release in recombinant Escherichia coli: a comparative analysis. Eng Life Sci. 2017;17:215–222. doi: 10.1002/elsc.201600168.

2. Chen Z-Y, Cao J, Xie L, Li X-F, Yu Z-H, Tong W-Y. Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli. Microb Biotechnol. 2014;7:360–370. doi: 10.1111/1751-7915.12127.