Chaperone protein system
To ensure that our growth factors can be correctly folded in L. lactis, we will introduce chaperone proteins into the environment. After reviewing the literature, we have selected the DnaK-DnaJ complex as the chaperone proteins for hGM-CSF, with the expression vector being pKJE7.
When DNA is transcribed and translated into a polypeptide chain, it first associates with DnaK, which is then detected and bound by DnaJ at hydrophobic amino acid residues, preventing unnecessary aggregation. After folding assistance, three potential outcomes can occur: successful folding, incomplete folding, or failure of folding leading to degradation. In cases of incomplete folding, the GroEL–GroES chaperone complex interacts with the polypeptide, attempting to refold it. This entire process constitutes a chaperone protein system, in which we predict that L. lactis is relatively deficient in DnaK and DnaJ. Therefore, the additional pKJE7 (BBa_K5361007) plasmid is employed in our experiments to enhance the folding success rate of hGM-CSF.
| item | number | name | description |
| 1 | BBa_K5361002 | rhGM-CSF | Growth factor |
| 2 | BBa_K5361001 | PelB-rhGM-CSF | Growth factor with secretion peptide PelB |
| 3 | BBa_K5361004 | Usp45-rhGM-CSF | Growth factor with secretion peptide Usp45 |
| 4 | BBa_J70004 | pUC57 | Vector |
| 5 | BBa_K5361003 | pUCIDT (Amp) Golden Gate | Vector |
| 6 | BBa_K5361005 | PelB-rhGM-CSF in pUCIDT (Amp) Golden Gate | BBa_K5361001 + BBa_K5361003 |
| 7 | BBa_K5361006 | Usp45-rhGM-CSF in pUCIDT (Amp) Golden Gate | BBa_K5361004 + BBa_K5361003 |
| 8 | BBa_K5361007 | pKJE7 | Chaperone protein set plasmid |
| 9 | BBa_K5361900 | pMG36E | Vector for L. lactis and E. coli |