Sumarry
We synthesized some model proteins, which have multiple disulfide bonds and show activity when a correct disulfide bond is formed. Some proteins could form a correct disulfide bond using GSSG (oxidizing agent) only. In most cases, isomerase, DsbC or PDI, is required for activity.
Materials and Methods
| Cell-free Protein Synthesis kit | PUREfrex® 2.1 (#PF213-0.25-EX) |
| Supplement | DsbC Set (#PF005-0.5-EX) |
| PDI Set (#PF006-0.5-EX) | |
| Incubation time | 4 hours |
| Temperature | 37 °C |
* GSSG oxidizes PDI and has a role to move the whole reaction solution in the oxidation direction.
* Ero1α is an enzyme that specifically oxidizes PDI and does NOT move the whole reaction solution in the oxidation direction.
Results
Plasminogen activator
| Organism | Homo sapiens |
| Synthesized region | 36Ser – 40Ile / 211Gly – 562Pro (+FLAG) |
| UniProt ID | P00750 |
| Length | 368 a.a. |
| Molecular weight | 41,072 Da |
| No. of disulfide bonds | 9 |
Acetylcholinesterase
| Organism | Homo sapiens |
| Synthesized region | 32Glu – 614Leu (+FLAG – His6) |
| UniProt ID | P22303 |
| Length | 602 a.a. |
| Molecular weight | 66,812 Da |
| No. of disulfide bonds | 4 (intermolecular: 1) |
Luciferase (Gaussia princeps)
| Organism | Gaussia princeps |
| Synthesized region | 18Lys – 185Asp (+FLAG-His6) |
| UniProt ID | Q9BLZ2 |
| Length | 187 a.a. |
| Molecular weight | 20,407 Da |
| No. of disulfide bonds | 5 |
Immunogloblin (Trastuzmab)
| Organism | humanized monoclonal antibody (IgG1 kappa) |
| Synthesized region | Heavy chain (HC): 1Gln – 451Lys (+FLAG) Light chain (LC): 1Asp – 214Cys |
| Length | HC: 462 a.a. / LC: 215 a.a. |
| Molecular weight | HC: 50,618 Da / LC: 23,573 Da |
| No. of disulfide bonds | 16 (intermolecular: 4) |
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