Question

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.

Answer 1

The designed protein that is expressed in E.coli is secreted outside of the cell for purification. In case of gram negative bacteria like E.coli the Sec or Tat pathways is used as secretion system to deliver the protein int the cytopasmic membrane or outside the cell. Here in this case, the designed protein is transported outside the cell and the protein folds after it has excited the cell, so Tat secretion system works best for this project as it primarily secretes folded proteins in contrast to Sec pathway, where the protein is secreted in unfolded state.

The Tat pathway of protein secretion consists of 2-3 subunits, TatA, TatB and TatC. In E.coli, the TatB and TatC bind the signal peptide of Tat secreted proteins and recruit TatA which forms the membrane spanning channel. The above designed protein is secreted outside the cell for E.coli, so the Tat system employs Type II secretion system. In gram negative bacteria like E.coli extracellular protein secretion is done in two stages. First the secreted protein is delivered to the periplasm via Tat pathway and secondly, it is transferred outside the cell via a second transport system which are six in number from Type I to Type VI that transports a specific subset of protein. The Type II secretion system here is operational which transport the folded protein outside the cell through a definite mechanism.

The Type II secretion system consist of 15 different proteins which are broken into four assemblies namely- (a) Outer membrane complex- It resides in the outer membrane where it forms a channel through which periplasmic proteins are translocated. This channel is composed of a multimeric protein called secretin. It has a long N terminus and extend to the inner membrane.

(b) Inner membrane platform- It is composed of multiple copies of atleast 4 proteins, embeded in the inner membrane and remain in contact with secretin. It plays an important role in the secretion process by communicating with the secretin, pseudopilus and the ATPase to coordinate the export of substrates.

(c) ATPase- It is located in the cytoplasm and provide energy to power the system.

(d) Pseudopilus- It is structurally similar to proteins that make the bacterial competence system.

The mechanism for secretion through Type II secretion system proposes that these pseudopili withdraw in order to push the folded protein substrate through the outer membrane channel. The above model is also referred as 'piston mode' in which, secretion competent proteins of the periplasm remain in contact with the periplasmic portion of secretin protein. this interaction is believed to stimulate the cytoplasmic ATPase to drive retraction of the Type II secretion system pseudopili, which push the folded protein through the secretin channel.