Parts

Parts

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.

Figure A-II: pKJE7 chaperone system plasmid.

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