Table 1 Selection of mtDod constructs used for expression studies are divided into two groups: mtDod-peptides (constructs with only short peptides fused to mtDod) and mtDod-proteins (constructs with domains or entire proteins fused to mtDod). mtDod(WT): wild type mtDod. seACP: Saccharopolyspora erythraea ACP, gene chlB2. msfGFP: monomeric superfolder green fluorescent protein29. For a full description of constructs, see Supplementary Table S1. The constructs mtDod-GSG-Lys and mtDod-PAS-Met have been used as control; MtDod-GSG-Lys was used for its flexible linker, and mtDod-PAS-Met for its rigid linker. The PAS linker were based on sequences (slight alterations) shown in ref30.

From: Dodecin as carrier protein for immunizations and bioengineering applications

Construct name

Linker system

Molar mass/Da

Expression state#

mtDod-peptides

 

mtDod(WT)

7,497

Soluble

mtDod-GSG-Lys

GSG

8,411

Soluble

mtDod-PAS-Met

PAS

8,876

Soluble

mtDod-SpyT

PASG

10,458

Soluble

SpyT-mtDod

GPAS

10,215

Soluble

mtDod-PAS2-SpyT

PAS2G

11,447

Soluble

SpyT-PAS2-mtDod

GPAS2

11,205

Soluble

SpyT-mtDod-SnpT

GPAS / PASG

13,142

Soluble

mtDod-proteins

mtDod-mmACP

PAS

17,994

Soluble

mtDod-mmACP-H8

PAS

19,334

Soluble

mtDod-msfGFP-H8

PAS

36,388

Soluble

mtDod-SpyC-H8*

PAS

22,413

Inclusion body

H8-SpyC-mtDod

PAS

22,072

Inclusion body

mtDod-SZ1**

PAS

14,232

Inclusion body

mtDod-SZ3**

PAS

13,396

Inclusion body

mtDod-seACP***

PAS

19,966

Inclusion body

Linker details

GSG:

GGGGSGGGG

PAS:

SPAAPAPASPAS

PASG:

SPAAPAPASPASGGSG

GPAS:

GGSGSPAAPAPASPAS

PAS2G:

SPAAPAPASPASPAPSAPAASPAAGGSG

GPAS2:

GGSGSPAAPAPASPASPAPSAPAASPAA

  1. #Describes whether the major fraction of the expressed construct is soluble or forms inclusion bodies. MtDod constructs with low solubility form often non-classical inclusion bodies (correctly folded protein)31, leading to their yellowish colouring (flavin binding). Since flavin binding only requires intact mtDod, it is possible that inclusion bodies are yellow although the protein cargo is misfolded.
  2. *MtDod-SpyC-H8 seems to be soluble in cellular environment but forms yellow aggregates after cell lysis.
  3. **SZ1-mtDod and SZ3-mtDod also formed inclusion bodies and behaved similarly as the C-terminal constructs (data not shown).
  4. ***MtDod-seACP could not be obtained in soluble form; under all applied refolding conditions yellow aggregate was formed.