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Fig. 4 | Microbial Cell Factories

Fig. 4

From: Protein purification strategies must consider downstream applications and individual biological characteristics

Fig. 4

Reconstruction of the disulfide bridge improves the folding and yield of soluble LLT1. a Gel filtration of wild-type recombinant soluble LLT1 (blue) and its C163S (orange) and H176C (green) mutants produced in HEK293T cell line. b Mass spectrometry analysis of disulfide bond pattern in wild-type LLT1 and its H176C mutant using samples from the peak at 16 ml position on SEC run shown in a corresponding to the LLT1 non-covalent dimer. The relative intensity of observed cystic peptides is shown. While in the H176C mutant both the expected two native disulfide bridges (Cys75-Cys86 and Cys103-Cys184) and the third reconstituted bond Cys163-Cys176 were formed (marked by asterisks), in the wild-type LLT1 the odd Cys163 residue paired randomly with other cysteines, leading to protein misfolding and aggregation. c Crystal structure of LLT1 (PDB 4QKI) non-covalent dimer (cyan and green) confirmed the expected disulfide bond pattern (in yellow with the reconstituted Cys163-Cys176 disulfide highlighted in red). (Original figure from Vaněk’s lab)

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