Overexpression of an archaeal protein in yeast: secretion bottleneck at the ERJason D Smith
Department of Chemical Engineering, University of Delaware, Newark 19716, USA
Biotechnol Bioeng 79:713-23. 2002
..Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields...
- Maximizing recovery of native protein from aggregates by optimizing pressure treatment
Brian G Lefebvre
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA
Biotechnol Prog 20:623-9. 2004
..Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields...
- Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate
Sara Lawrence Powers
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
Extremophiles 11:179-89. 2007
..The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding...
Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspikeBrian G Lefebvre
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
Protein Sci 13:1538-46. 2004
..Taken together, these results suggest that E196K has a primary defect in formation of the beta-helix during monomer collapse, while G244R is primarily an assembly defect...
- Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast
Ping Xu
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA
Biotechnol Bioeng 104:20-9. 2009
..This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties...
- PDI improves secretion of redox-inactive beta-glucosidase
Sara Lawrence Powers
Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, Delaware 19716, USA
Biotechnol Prog 23:364-9. 2007
..These results suggest that, unexpectedly, PDI acts in a chaperone-like capacity or possibly cooperates with the cell's folding or degradation mechanisms regardless of whether the protein is redox-active...
- A top-down approach to mechanistic biological modeling: application to the single-chain antibody folding pathway
Scott Hildebrandt
Department of Chemical Engineering, Department of Computer Science, University of California, Santa Barbara, California, USA
Biophys J 95:3535-58. 2008
..It also prescribed modification of the guiding hypothesis to capture BiP and PDI synergy...
- Heterologous GPCR expression: a bottleneck to obtaining crystal structures
Emily C McCusker
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19711, USA
Biotechnol Prog 23:540-7. 2007
..Developing reliable expression techniques for GPCRs is still a major priority for the structural characterization of GPCRs...
- Three amino acids that are critical to formation and stability of the P22 tailspike trimer
Matthew J Gage
259 Colburn Laboratory, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
Protein Sci 14:2333-43. 2005
..We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation...
- Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli
Steven E Bane
Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA
Protein Expr Purif 52:348-55. 2007
..Thus this protein is suitable for additional biophysical characterization and refolding studies...
- Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast
Jason D Smith
Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, Delaware 19716, USA
Biotechnol Bioeng 85:340-50. 2004
..We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process...
- Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding
Ping Xu
259 Colburn Laboratory, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
Metab Eng 7:269-79. 2005
..Free BiP is released when scFv is folded, stabilizing Ire1p, and leading to the reduced UPR...
- Exceptional total and functional yields of the human adenosine (A2a) receptor expressed in the yeast Saccharomyces cerevisiae
Ronald T Niebauer
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
Protein Expr Purif 46:204-11. 2006
..Importantly, the amount of functionally expressed A2a-GFP per culture ( approximately 4 mg/L) is among the highest reported for any GPCR in any expression system...
- Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein
Brenda L Danek
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA
Biophys J 85:3237-47. 2003
..Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike...
- Refolding of G protein alpha subunits from inclusion bodies expressed in Escherichia coli
Emily McCusker
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
Protein Expr Purif 58:342-55. 2008
..We conclude that the refolded G(ialpha) protein has a native-like secondary structure, but is predominantly in a molten globular state...
- Decreases in yeast expression yields of the human adenosine A2a receptor are a result of translational or post-translational events
Ronald T Niebauer
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
Protein Expr Purif 37:134-43. 2004
..Taken together, these results suggest that heterologous expression of GPCRs is limited by a translational or post-translational bottleneck that is unique from expression limitations seen for soluble proteins...
Computational design and biophysical characterization of aggregation-resistant point mutations for γD crystallin illustrate a balance of conformational stability and intrinsic aggregation propensityErinc Sahin
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, United States
Biochemistry 50:628-39. 2011
..Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q...
- C-terminal hydrophobic interactions play a critical role in oligomeric assembly of the P22 tailspike trimer
Matthew J Gage
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA
Protein Sci 12:2732-47. 2003
..Taken together, these results suggest that hydrophobic interactions between C-terminal regions of P22 tailspike monomers play a critical role in the assembly of the P22 tailspike trimer...
- Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast
James A Butz
Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, DE 19716, USA
Biotechnol Bioeng 84:292-304. 2003
..Taken together, these results indicate that an ER-folding bottleneck does not limit the expression of the mammalian receptors in yeast...
- Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS
Junghwa Kim
Department of Chemical Engineering, University of Delaware, Newark 19716, USA
Protein Sci 15:1791-3. 2006
..This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers...
- Pressure treatment of tailspike aggregates rapidly produces on-pathway folding intermediates
Brian G Lefebvre
Department of Chemical Engineering, 259 Colburn Laboratory, University of Delaware, Newark, Delaware 19716, USA
Biotechnol Bioeng 82:595-604. 2003
..Circular dichroism and high-pressure intrinsic tryptophan fluorescence measurements support the model that a structured intermediate is formed in a rapid manner under high pressure from a pressure-sensitive aggregate population...
- Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae
Kenneth J Kauffman
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA
Biotechnol Prog 18:942-50. 2002
..Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels...
