title: Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system.
abstract: The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.
authors.affiliation: Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Welsh
authors.forename: Jeanne
authors.initials: J
authors.lastname: Bonomo
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002474:Cell-Free System; D000091342:Endoplasmic Reticulum Chaperone BiP; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014175:Translating
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase