1. iGEM Meeting – 5.26.08 Thomas Graham

2. Option 1: E. coli Gram-negative = two membranes

3. Type I secretion in E. coli Problems: Hemolysin subunits are limiting  overexpression of all of them required Even with overexpression of all subunits, the system is easily saturated.

4. Expression of Type I-secreted proteins

5. Type II secretion in E. coli Most endogenous proteins transported by this mechanism localize to the periplasm or inner membrane. Two pathways for secretion across the outer membrane: leakage or main terminal branch (MTB) mechanism.  Unfortunately, endogenous MTB proteins are not expressed at high levels.

6. E. coli summary Most secreted proteins are periplasmic or membrane associated. Extracellular secretion is very problematic – the bacterial secretion apparatus is not optimized for high-level secretion.

7. Option 2: Bacillus sp. Gram positive = one membrane Has the ability to sporulate (might be useful for long-term storage) Not as easily to transform as E. coli

8. B. subtilis expression/secretion system Lots of endogenous proteins secreted (e.g. subtilisin) Protein of interest can be fused to a short signal peptide (e.g. sacB signal peptide) Protease-deficient strain WB800 doesn’t chew up your overexpressed protein Production levels of up to 3 g/liter of culture—but this is not always the case. Used for commercial production of enzymes, e.g. for laundry detergents Various inducible promoter systems available

9. Bacilli

10. Option 3: Caulobacter crescentus Gram-negative like E. coli, but secretes large amounts of endogenous RsaA protein through type I secretion. Fusion of RsaA signal sequence to protein targets it for extracellular secretion Size limit ~450 aa. Not very widely used for protein production Can survive in nutrient-poor environments – normally found in fresh water and soil We have a C. crescentus expert on faculty (Sean Crosson) Organism is most popular for mathematical modeling studies

11. Caulobacter

12. Other options Pseudomonas fluorescens Ralstonia eutropha Neither is commonly used, but both have yielded impressive amounts of protein

13. Things to worry about… Protein toxicity to the host Protein folding –SRP mechanism – concomitant translation and secretion –Sec mechanism – uncoupled translation/secretion; requires unfolding/refolding –Both mechanisms require folding of the protein in the extracellular environment, which can in some cases be a problem Disulfide bond formation (is this of concern to us?) Tyrosinase is bigger and will probably be secreted less efficiently Difficult to transform Bacillus