PATh :.: Process and Product Applied Thermodynamics ...



Table A.1 E. coli strains used in this work.

|E. coli strain |Genotype |Selection marker |Manufacturer |

|DH5α1 |[deoR endA1 gyrA96 hsdR17 Δ(lac)U169 recA1 relA1 supE44 thi-1 (Φ 80 |- |Novagen™ |

| |lacZΔM15)] | | |

|AD4942 |[Δ(araABC-leu)7697 ΔlacX74 ΔmalF3 ΔphoAPvuII phoR trxB ::Kanr |Kan |Novagen™ |

| |F'[lacIqlacZΔM15proAB+] | | |

|BL21(DE3)3 |[F– ompT hsdSB(rB–, mB–) gal dcm (DE3)] |- |Life Technologies™ |

|BL21 CodonPlus(DE3)4 |[RIPL: endA gal ompT hsdSB Dcm+ Hte Tetr (pACYC-RIL argU ileY leuW Cam]r |Cam and Strep |Agilent Technologies |

| |(DE3) | | |

|Tuner™ (DE3) |gal hsdSB lacY1 ompT (DE3) | |Novagen™ |

|BL21pLyss(DE3)5 |F–, ompT, hsdSB (rB–, mB–), dcm, gal, λ(DE3), pLysS, Cmr |Cam |Promega™ |

|C43 (DE3)6 |[F – ompT hsdSB (rB- mB-) gal dcm (DE3)] |- |Lucigen ™ |

|Rosetta™(DE3)7 |F– ompT hsdSB(rB – mB –) gal dcm (DE3) pRARE2 (CamR) |Cam |Novagen™ |

|Origami(DE3)8 |1∆(ara–leu)7697 ∆lacX74 ∆phoA PvuII phoR araD139 ahpC galE galK rpsL F'[lac+|Cam, Strep and Tetra |Novagen™ |

| |lacI q pro] (DE3) gor522::Tn10 trxB | | |

- = no selective marker, Kan = kanamycin, Cam = chloramphenicol, Tetra = tetracycline, Strep = streptomycin. Main reason to use this strain: 1 Cloning vectors; 2 (trxB) mutants that enable disulfide bond formation in the E.coli cytoplasm expressed proteins; 3 Expression of cytoplasmic proteins and deficient in both lon and ompT proteases; 4 extra copies of genes that encode the tRNAs that most frequently limit translation of heterologous proteins in E. coli; 5 high-stringency expression host due to expression of T7 lysozyme; 6 Effective in expressing toxic and membrane proteins; 7 Expresses six rare tRNAs that most frequently limit translation of heterologous proteins in E. coli; 8 trxB/gor mutant, greatly facilitates cytoplasmic disulfide bond formation.

Table A.2 Screening of expression conditions.

|Bacteria strain |pET15b |pET22b |pET28a+SUMO |T °C |

|ASP3_1-362 |ASP3_32-362 |ASP3opt_26-362 |ASP3_26-362 |ASP3_26-362 | | |AD494 |I |I |I |I |I |I |I |I |I |. |. |. |. |. |. |20 | | |I |I |I |I |I |I |. |. |. |. |. |. |. |. |. |37 | |BL21(DE3) |I |I |I |I |I |I |I |I |I |. |. |. |I |I |I |20 | | |I |I |I |I |I |I |. |. |. |I |I |I |. |. |. |37 | |C43(DE3) |I |I |I |I |I |I |0 |0 |0 |. |. |. |. |. |. |20 | | |I |I |I |I |I |I |. |. |. |0 |0 |0 |. |. |. |37 | |BL21 CodonPlus (DE3) |. |. |. |. |. |. |. |. |. |. |. |. |. |. |. |20 | | |I |I |I |I |I |I |. |. |. |. |. |. |. |. |. |37 | |Tuner (DE3) |I |I |I |I |I |I |I |I |I |. |. |. |. |. |. |20 | | |I |I |I |I |I |I |. |. |. |I |I |I |. |. |. |37 | |BL21pLysS (DE3) |. |. |. |. |. |. |. |. |. |. |. |. |0 |0 |0 |20 | | |. |. |. |. |. |. |. |. |. |0 |0 |0 |. |. |. |37 | |Rosetta (DE3) |. |. |. |. |. |. |. |. |. |. |. |. |0 |0 |0 |20 | | |. |. |. |. |. |. |. |. |. |. |. |. |. |. |. |37 | |Origami (DE3) |. |. |. |. |. |. |I |I |I |. |. |. |. |. |. |20 | | |. |. |. |. |. |. |. |. |. |0 |0 |0 |. |. |. |37 | |IPTG (mM) |0.1 |0.5 |1.0 |0.1 |0.5 |1.0 |0. 01 |0.1 |1.0 |0.1 |0.5 |1.0 |0.01 |0.1 |1.0 | | |I = Insoluble, S = Soluble, 0 = Do not express, = Not tested.

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