CHE.496/2009/Schedule/Oral presentations of system designs/Group 1

Recombinant Protein Secretion in Escherichia coli

 * The gram negative bacteria secrete proteins across two membranes that differ in composition and function.
 * (Cytoplasm --> IM --> Periplasm --> OM)
 * Protein secretion into the culture medium is also done by leakage of periplasmic contents

Secretion Pathways:
 * Type 1 secretion
 * Type 2 secretion

1)	Type I secretion mechanism
 * Transport proteins in one step across the two cellular membranes without a periplasmic intermediate
 * For secretion of high molecular weight toxins and exoenzymes. The pathway secretes proteins ranging from 50-4000 amino acids and globular proteins up to 200 amino acids.
 * Consists of: 2 inner membrane proteins (HlyB and HlyD), an endogenous outer membrane protein, TolC and Type I transporter (most popular-(Hly A transporter)
 * Tag for the recombinant protein is a C-terminal Hly A signal sequence
 * How it works: The two proteins (HlyB and HlyD) form a complex and bind to the recombinant protein with the C-terminal HlyA signal sequence and ATP in the cytoplasm. The TolC trimer binds to the complex forming a channel connecting both cellular membranes. The bacterial protein TolC assembles into an alpha helical trans-perisplasmic tunnel embedded in the outer membrane by a beta barrel channel.
 * ATP is hydrolyzed by HlyB in order to transport the protein through the channel. After translocation, TolC separates from the complex and disconnects the membrane.
 * The channel is 3.5 nm (diameter) and 14 nm (length).
 * Type I secretion can export the target protein to the culture medium.

Draw Backs of Type I secretion: 1)	The signal sequence is attached to the secrete peptide, needs additional cleavage step 2)	Coexpression of the components of this system is needed to increase the transport capacity. This can cause a bottleneck problem with congestion of proteins due to low protein translation rates.

2)	Type II secretion mechanism a)	SecB-dependent b)	Signal Recognition Particle (SRP) c)	Twin-arginine Translocation (TAT)
 * Two step process for extracellular secretion of proteins mediated by periplasmic translocation
 * Three pathways can be used for the first step (secreting across bacterial cytoplasmic membrane) which include:


 * The second step uses a protein called secreton for translocation across the outer membrane

1st Step:

>	SecB-dependent translocation •	Ribosome-associated chains of secreted proteins bind trigger factor •	SecB protein recognizes it and targets it to the membrane bound SecA •	At the translocation point, a group of proteins (Sec Y, SecE, SecG) form a complex that threads the protein using ATP through the translocation channel •	ATP hydrolysis releases the protein from SecA into the channel. ADP is released and SecA deinserts from the membrane and is exchanged for a cytosolic SecA. The continuous deinsertion and insertion of SecA promote protein translocation through the channel. •	The secreted protein has an amino-terminal signal peptide that works as a targeting/recognition signal •	Signal peptide is 18-30 amino acid residues long, +ve charged, central hydrophobic core and a polar cleavage region •	Chaperone SecB prevents pre-mature folding of the protein

Draw Backs of SecD Pathway: 1)	SecA expression is down-regulated by the binding of SecA to its own mRNA (control mechanism) 2)	SecA translation is regulated by the product of a cotranscribed upstream gene, secM which monitors E.coli secretion proficiency. When SecM secretion is limiting, ribosomes translating secM expose the secA ribosome binding site in secM-secA mRNA thereby stimulating secA translation. When the cell has excess protein secretion capacity, translocation of SecM is efficient and secA translation is repressed. 3)	System cannot transport folded proteins

>	SRP pathway •	N-terminal signal sequence binds to the Signal Recognition Particle (SRP) •	The hydrophobicity of the signal sequence on the mRNA determines the rate of translocation •	FtsY and Ffh found in the cytoplasm and membrane interact with the SRP complex. •	GTPase activites of FtsY and Ffh are stimulated releasing the chain to the translocation site •	Protein YidC allows for the lateral diffusion of the proteins to the periplasm

>	TAT pathway •	Twin-arginine translocation system- protein transported by it have two consecutive and highly conserved arginine residues in their leader peptides •	Protein is fully synthesized and folded in the cytoplasm where it binds to specific cofactors •	TatC in the TatBC complex recognize the signal peptide •	Signal peptide binding promotes association of the complex with TatA oligomers by the proton motive force (PMF) driving translocation. •	TatA forms the transport channel •	In the periplasm, the protein is folded and develops its tertiary and quaternary structures. •	TAT signal peptides have three regions: (i) +ve charged (n-region), (ii) hydrophobic (h-region), (iii) cleavage site (c-region). The TAT signal peptides are 38 amino acids in length

Drawbacks of TAT pathway:

1)	Less efficient, slower than the Sec pathway 2)	Secretion mechanism is rapidly saturated 3)	High energy cost of translocation

2nd Step (Extracellular Secretion):

>	Secreton -use of leaky strains -cell membrane permeabilization -coexpression of release proteins
 * (12-16 proteins) secretion machinery
 * Extracellular release through:
 * Uses main terminal branch (MTB) of the general secretory pathway
 * E.coli does not express its genes coding for MTB components under normal lab conditions, so some secreton components are needed to be overexpressed or the environmental conditions need to be optimized to allow for their expression

Periplasmic Leakage: -cell division, leakage of periplasmic contents occurs before the formation of the outer membranes -accumulation of protein in the periplasm cause an increase in osmotic pressure diving transport across the outer membrane -recombinant protein production can induce perturbations on the membrane increasing its selective permeability facilitating leakage -periplasmic secretion can cause cell lysis resulting in the release of periplasmic content •	Strategies to increase the permeability of the outer membrane:
 * Several Causes:
 * a)	Mechanical (ultrasound)
 * b)	Chemical (addition of magnesium, calcium, EDTA, glycine and Triton X-100)
 * c)	Enzymatic (lysozyme) treatments
 * d)	Chemical parameters (temperature, culture medium composition, pH, aeration)
 * Leaky Strain-mutant that displays disturbances in the synthesis of outer membrane components
 * Use of growth-arrested metabolically active quiescent cells- protein can be expressed in an environment where resource competition for transcription, translation and secretion is reduced.
 * Co-expression strategies