(provided by application): The long-term objective of this program is to improve our understanding of the protein folding process for secreted, complex mammalian proteins and to produce better technology for their production.
The specific aims of this proposal are: A. To identify and produce a fusion protein with full BiP activity that is localized near the exit port of a prokaryotic ribosome, and B. To determine the effect of the localized BiP activity on the expression and folding of huGCSF and a Fab antibody fragment. Using huGCSF, and a murine Fab antibody fragment as model proteins; a prokaryotic system will be established to mimic essential features of eukaryotic protein secretion into the endoplasmic reticulum. Early results show that a new E.coli cell-free system: a) supports prolonged protein synthesis, b) provides a stable chemical environment, c) proceeds with eukaryotic or slower translation rates, and d) provides effective disulfide bond formation and isomerization. Trigger factor, the prokaryotic ribosome-associated chaperone, inhibits cell-free expression of huGCSF, presumably by slowing translation through ineffective chaperone interactions. Trigger factor will be replaced at the exit port of the ribosome with the prokaryotic, proximal chaperone, BiP. BiP will be augmented with ERdj3 to stimulate BiP association with target sequences and its ATPase activity and with BAP, BiP's nucleotide exchange catalyst. Reagents will be carefully analyzed to insure activity. Target protein expression will be optimized, and a variety of radioactive labeling and Immuno-chemical techniques coupled with high-resolution polysome analysis will be used to assess the precise effect of the proximal Chaperones. If successful, the project will produce new knowledge about eukaryotic protein folding and will also produce improved technology for the production of important protein therapeutics. ? ? ?
| Welsh, John P; Bonomo, Jeanne; Swartz, James R (2011) Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system. Biotechnol Bioeng 108:1739-48 |
| Bonomo, Jeanne; Welsh, John P; Manthiram, Karthish et al. (2010) Comparing the functional properties of the Hsp70 chaperones, DnaK and BiP. Biophys Chem 149:58-66 |
