The Gatekeeper A new secreting method for E. coli

Introduction Numerous useful proteins are produced as recombinant proteins because of the simplicity and a cheaper process compared to chemical synthesis. Usual hosts are for examples S. cerevisiae, tobacco cells… The most used host is E. coli because of these several advantages: • Well-known genetics • Easily grown (possibility of mass production in fermenter) • High expression (several grams per liter) • Low production cost There are some drawbacks as well such as the lack of post-translational modifications permitting to obtain a human compatible protein.

Students : Dekoninck, K., Devreux, B., Fivet, D., Gauchet, A., Godeaux, A., Godefroi, G., Gossart, N., Guzman Espinola, A. J., Masscheleyn, P.-A., Tyckaert, F., Vellings, M. Academics : Hallet, B. & Soumillon, P.

As Escherichia coli don’t secrete easily a high level of recombinant protein into the extracellular medium, the release of the synthetized protein requires a lytic step. Such step requires purification, solubilisation and implies some renaturations problems.

Recombinant proteins strategies Domain

Pro Simpler purification Less extensive proteolysis Improved folding N-terminus authenticity

Cons Possible formation of inclusion body Signal not always facilitate exported

Periplasm Cytosol

Cytosol inclusion body

Extracellular

Higher yield Simpler plasmid construction

Facile isolation Protection from protease Protein inactive

Less extensive proteolysis Simpler purification Improved folding N-terminal authenticity

Université Catholique de Louvain

Our Project The aim of our project is to make E. coli able to secrete recombinant proteins outside the periplasm. To do so, we will use transformed E. coli expressing an outer membrane phage protein (pIV), a channel that can be opened or closed under certain conditions. By mutating certain regions of pIV using a random mutagenesis method, we hope to create a regulable mutant where the release of recombinant proteins can be activated in the presence or absence of a specific inductor.

Disulfur bond formation disfavored N-terminal authenticity Proteolysis More complex purification

• • • •

Inductor + / Inductor -

Inductor - / Inductor +

• •

Protein misfolding Lower yield Higher cost of goods

pIV secretin Secretin from the filamentous bacteriophage f1. interacting with pI and pIX in the inner membrane to permit the phage assembly and extrusion. Radially symmetrical 14-meric complex. Barrel-like structure comprised of three stacked rings defining a discontinuous pore in the center. The pore diameter ranges from 6 nm to 8.8 nm. Such a large channel could compromise the integrity of the outer membrane.

 In physiologic conditions, PIV opening is tightly regulated and happens only during the phage extrusion.

Usually no secretion More complex purification (than in cytosol)

Capsid proteins

(Adapted from Hanning & Makrides,1998)

Viral ssDNA

 In the industry, secretion is the best way to recover the protein but the yield is very low. Usually the protein is secreted in the periplasm and then the cell is lysed.

Base of the experiment C

(Adapted from Rakonjac et al., 2011)

Random mutagenesis

Outer membrane 1. Cloning

E. coli ΔlamB

2. Mutagenesis

N

LamB is the unique specific maltose and maltodextrins transporter of E. coli. Periplasm

∆lamB strain is unable to grow in restrictive medium with pentamaltose as the only carbon source.

ABres : antibiotic resistance selection gene Pro : propionate inducible promoter pIV : wt porin gene pIV* : mutant porin gene

N : N-terminal domain C : C-terminal domain N0 : most N-terminal subdomain (FpvA-like domain : domain of the outer membrane Ferripyoverdine receptor) N3 : N-terminal KH-fold subdomain [KH-domain = binding site with RNA/ssDNA

GATE1 : clustered region spanned 39 residues [a.a.] that resulted several leaky phenotype after mutation GATE2 : clustered region spanned 14 residues [a.a.] that resulted several leaky phenotype after mutation (Spagnuolo et al., 2010)

Spagnuolo et al. (2010) have defined two gating regions : GATE1 and GATE2, where amino acids modifications led to “leaky” mutants. These “leaky” mutants exhibed an outer membrane permeable to vancomycin and maltooligosaccharides. We will use this knowledge to find a regulable mutant by random mutagenesis of these gating regions.

3. Gibson assembly

1.

Cloning the pIV wt gene in a vector under a propionate tightly regulable inducible promoter.

2.

Insertion of a random mutated region using an external degenerated primer.

3.

Recircularization of the vector following a Gibson assembly method.

Vancomycine Vancomycine is an antibiotic of the family of the glycopeptids. It inhibits the synthesis of the peptidoglycan of the cell wall. This antibiotic can not diffuse through the external membrane.

Regulable “leaky“ mutants

OUR GOAL : create a “leaky“ mutant regulable under specific conditions such as variation of temperature, pH, etc. or the addition of a chemical inductor (cationic agent, cofactor…) Phase 1 : Creation and obtention of regulable “leaky“ mutants • Use of a ∆lamB strain of E. coli • Transformation by our biobrick of pIV* • Growth of E. coli under different specific conditions

Tag specific opening OUR GOAL : create a pIV that is opened by the production of a tagged recombinant protein Phase 1 and 2 at the same time

∆lamB E. coli

Growth on a rich medium

Induction of pIV*

 Identification of regulable leaky mutants under specific condition  Determination of closing inductor or opening inductor

Induction of the tag-protein

Phase 2 : Secretion efficiency assay • Transformation by vector containing a gene of a recombinant protein targeting to the periplasm and easily detectable • Use of the previously determined specific conditions for the induction of pIV* and induction of the reporter protein • Characterization of each selected mutant If we succeed in our project, we will have a new kind of pIV allowing E. coli to secrete recombinant proteins from the periplasm to the external environment thanks to a specific determined induction condition.

Transformation by our biobrick

Transformation by the vector containing the tag-protein

Change of medium to a restrictive medium

No change of medium, but add of vancomycine

If growth, opened porin = interaction tag-pIV*

If growth, closed porin = no interaction tag-pIV*

If we succeed in our project, we will have a new kind of pIV permitting to E. coli to secrete recombinant proteins specifically tagged from the periplasm to the external environment.

Perspectives

No induction of the tag-protein

Selection pIV* regulable by interaction tag-pIV*

CMFR

These results could be exploited to develop a new kind of cells culture in bioreactors or to improve some of them already existing . For instance, we can imagine using these mutants to rise the yields of a continuous culture (CMFR) : the ability to secrete the proteins of interest out of the cells may allow them to perform several production cycles in a row. Another interesting trail is the case of the production of a toxic protein for the cell : if this one can excrete the protein as it is produced, we can imagine reaching higher concentrations in the medium without killing the cells. One more improvement these mutants could bring is the reduction of the downtime in some processes : cells lysis step could be skipped as the product is dissolved in the medium and mostly, increase the purity et simplify the purification steps. We think our project could interest bioprocess companies to produce , for example, insulin, human growth hormone (HGH), interferon, factor VIII, etc.

References

Hannig, G., et S. C. Makrides. 1998. « Strategies for Optimizing Heterologous Protein Expression in Escherichia Coli ». Trends in Biotechnology 16 (2): 54-60. Rakonjac, Jasna, Nicholas J. Bennett, Julian Spagnuolo, Dragana Gagic, et Marjorie Russel. 2011. « Filamentous Bacteriophage: Biology, Phage Display and Nanotechnology Applications ». Current Issues in Molecular Biology 13 (2): 51-76. Spagnuolo, Julian, Natacha Opalka, Wesley X. Wen, Dragana Gagic, Elodie Chabaud, Pierdomenico Bellini, Matthew D. Bennett, et al. 2010. « Identification of the Gate Regions in the Primary Structure of the Secretin pIV ». Molecular Microbiology 76 (1): 133-150. doi:10.1111/j.1365-2958.2010.07085.x.