Decreased Secretion and Unfolded Protein Response Up-regulation Are Correlated with Intracellular Retention for Single-chain Antibody Variants Produced in Yeast

Overview

Journal Biotechnol Bioeng

Publisher Wiley

Specialty Biochemistry

Date 2009 May 6

PMID 19415776

Citations 5

Authors

Ping Xu

Anne Skaja Robinson

Affiliations

Soon will be listed here.

Abstract

Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the UPR, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4-4-20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the UPR. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. 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.

Citing Articles

Novel xylose transporter Cs4130 expands the sugar uptake repertoire in recombinant strains at high xylose concentrations.

Bueno J, Borelli G, Correa T, Fiamenghi M, Jose J, de Carvalho M Biotechnol Biofuels. 2020; 13:145.

PMID: 32818042 PMC: 7427733. DOI: 10.1186/s13068-020-01782-0.

Filamentous fungi-like secretory pathway strayed in a yeast system: peculiarities of Yarrowia lipolytica secretory pathway underlying its extraordinary performance.

Celinska E, Nicaud J Appl Microbiol Biotechnol. 2018; 103(1):39-52.

PMID: 30353423 PMC: 6311201. DOI: 10.1007/s00253-018-9450-2.

Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris.

Li C, Lin Y, Zheng X, Pang N, Liao X, Liu X BMC Biotechnol. 2015; 15:88.

PMID: 26410558 PMC: 4584009. DOI: 10.1186/s12896-015-0204-2.

Recombinant antibody production in Arabidopsis seeds triggers an unfolded protein response.

De Wilde K, De Buck S, Vanneste K, Depicker A Plant Physiol. 2012; 161(2):1021-33.

PMID: 23188806 PMC: 3561000. DOI: 10.1104/pp.112.209718.

Decreased secretion and unfolded protein response upregulation.

Young C, Yuraszeck T, Robinson A Methods Enzymol. 2011; 491:235-60.

PMID: 21329804 PMC: 4561176. DOI: 10.1016/B978-0-12-385928-0.00014-6.

References

Xu P, Raden D, Doyle 3rd F, Robinson A . Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding. Metab Eng. 2005; 7(4):269-79. DOI: 10.1016/j.ymben.2005.04.002. View

Knarr G, Gething M, Modrow S, Buchner J . BiP binding sequences in antibodies. J Biol Chem. 1995; 270(46):27589-94. DOI: 10.1074/jbc.270.46.27589. View

Feldhaus M, Siegel R . Yeast display of antibody fragments: a discovery and characterization platform. J Immunol Methods. 2004; 290(1-2):69-80. DOI: 10.1016/j.jim.2004.04.009. View

Whitlow M, Howard A, Wood J, Voss Jr E, HARDMAN K . 1.85 A structure of anti-fluorescein 4-4-20 Fab. Protein Eng. 1995; 8(8):749-61. DOI: 10.1093/protein/8.8.749. View

Takenaka I, Leung S, McAndrew S, Brown J, Hightower L . Hsc70-binding peptides selected from a phage display peptide library that resemble organellar targeting sequences. J Biol Chem. 1995; 270(34):19839-44. DOI: 10.1074/jbc.270.34.19839. View

Holliger P, Hudson P . Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005; 23(9):1126-36. DOI: 10.1038/nbt1142. View

Walsh G . Biopharmaceutical benchmarks--2003. Nat Biotechnol. 2003; 21(8):865-70. DOI: 10.1038/nbt0803-865. View

Spear E, Ng D . The unfolded protein response: no longer just a special teams player. Traffic. 2001; 2(8):515-23. DOI: 10.1034/j.1600-0854.2001.20801.x. View

Credle J, Finer-Moore J, Papa F, Stroud R, Walter P . On the mechanism of sensing unfolded protein in the endoplasmic reticulum. Proc Natl Acad Sci U S A. 2005; 102(52):18773-84. PMC: 1316886. DOI: 10.1073/pnas.0509487102. View

10.

Huang D, Gore P, Shusta E . Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss. Biotechnol Bioeng. 2008; 101(6):1264-75. DOI: 10.1002/bit.22019. View

11.

Rudiger S, Buchberger A, Bukau B . Interaction of Hsp70 chaperones with substrates. Nat Struct Biol. 1997; 4(5):342-9. DOI: 10.1038/nsb0597-342. View

12.

Sorgjerd K, Ghafouri B, Jonsson B, Kelly J, Blond S, Hammarstrom P . Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates amyloidogenesis. J Mol Biol. 2005; 356(2):469-82. DOI: 10.1016/j.jmb.2005.11.051. View

13.

Knarr G, Modrow S, Todd A, Gething M, Buchner J . BiP-binding sequences in HIV gp160. Implications for the binding specificity of bip. J Biol Chem. 1999; 274(42):29850-7. DOI: 10.1074/jbc.274.42.29850. View

14.

DAVIS D, Khurana R, Meredith S, Stevens F, Argon Y . Mapping the major interaction between binding protein and Ig light chains to sites within the variable domain. J Immunol. 1999; 163(7):3842-50. View

15.

Travers K, Patil C, Wodicka L, Lockhart D, Weissman J, Walter P . Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 2000; 101(3):249-58. DOI: 10.1016/s0092-8674(00)80835-1. View

16.

Shusta E, Raines R, Pluckthun A, Wittrup K . Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat Biotechnol. 1998; 16(8):773-7. DOI: 10.1038/nbt0898-773. View

17.

Gething M, Buchner J, Fourie A, Knarr G, Modrow S, NANU L . Binding sites for Hsp70 molecular chaperones in natural proteins. Cold Spring Harb Symp Quant Biol. 1995; 60:417-28. DOI: 10.1101/sqb.1995.060.01.046. View

18.

Kimata Y, Kimata Y, Shimizu Y, Abe H, Farcasanu I, Takeuchi M . Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins. Mol Biol Cell. 2003; 14(6):2559-69. PMC: 194903. DOI: 10.1091/mbc.e02-11-0708. View

19.

Cwirla S, Dower W, Lipshutz R, Sprang S, Sambrook J, Gething M . Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell. 1993; 75(4):717-28. DOI: 10.1016/0092-8674(93)90492-9. View

20.

Robinson A, Bockhaus J, Voegler A, Wittrup K . Reduction of BiP levels decreases heterologous protein secretion in Saccharomyces cerevisiae. J Biol Chem. 1996; 271(17):10017-22. DOI: 10.1074/jbc.271.17.10017. View