Abstract
Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the σ32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that σ32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor σ32. Manipulation of the σ32 regulon might provide a general mechanism for improving recombinant protein solubility.
Similar content being viewed by others
References
Jana S, Deb JK (2005) Appl Microbiol Biotechnol 67:289–298
Sorensen HP, Mortensen KK (2005) Microb Cell Fact 4:1–8
Baneyx F, Mujacic M (2004) Nature Biotechnol 22:1399–1408
Hoffmann F, Rinas U (2004) Adv Biochem Eng Biotechnol 89:143–161
Deuerling E, Schulze-Specking A, Tomoyasu T, Mogk A, Bukau B (1999) Nature 400:693–696
Teter SA, Houry WA, Ang D, Tradler T, Rockabrand D, Fischer G, Blum P, Georgopoulos C, Hartl FU (1999) Cell 97:755–765
Ewalt KL, Hendrick JP, Houry WA, Hartl FU (1997) Cell 90:491–500
Allen SP, Polazzi JO, Gierse JK, Easton AM (1992) J Bacteriol 174:6938–6947
Laskowska E, Wawrzynow A, Taylor A (1996) Biochimie 78:117–122
Zolkiewski M (1999) J Biol Chem 274:28083–28086
Grossman AD, Straus DB, Walter WA, Gross CA (1987) Genes Dev 1:179–184
Zhao K, Liu M, Burgess RR (2005) J Biol Chem 280:17758–17768
Thomas JG, Baneyx F (1996) J Biol Chem 271:11141–11147
Nishhara K, Kanemori M, Yanagi H, Yura T (2000) Appl Environ Microbiol 66:884–889
Chen J, Acton TB, Basu SK, Montelione GT, Inouye M (2002) J Mol Microbiol Biotechnol 4:519–524
Han MJ, Park SJ, Park TJ, Lee SY (2004) Biotechnol Bioeng 88:426–436
de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B (2007) E. coli. BMC Biotechnol 12:32
Rinas U, Hoffmann F, Betiku E, Estape D, Marten S (2007) J Biotechnol 127:244–257
Studier FW, Moffatt BA (1986) J Mol Biol 189:113–130
Smith HE, Ward S (1998) J Mol Biol 279:605–619
Studier FW (1991) J Mol Biol 219:37–44
Blackwell JR, Horgan R (1991) FEBS Letters 295:10–12
Lesley SA, Graziano J, Cho CY, Knuth MW, Klock HE (2002) PRotein Eng 15:153–160
Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado0Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) Science 277:1453–1474
Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA (2006) Genes Dev 20:1776–1789
Bullock TL, Roberts TM, Stewart M (1996) J Mol Biol 263:284–296
Hengge-Aronis R (1996) Mol Microbiol 21:887–893
Gamer J, Bujard H, Bukau B (1992) Cell 69:833–842
Tomoyasu T, Ogura T, Tatsuta T, Bukau (1998) Mol Microbiol 30:567–581
Guisbert E, Herman C, Lu CZ, Gross CA (2004) coli Genes Dev 18:2812–2821
Straus D, Walter W, Gross CA (1990) Genes Dev 4:2202–2209
Acknowledgements
We would like to thank Osnat Herzberg for providing expression plasmids for induction of soluble and insoluble proteins, Marc Salit for assistance in isolating some of the RNA samples, and Alvaro Godinez of the UMBI Microarray Core Facility for performing the microarray screens. We thank various members of the research groups of John Moult, Osnat Herzberg, and Fred Schwarz for fruitful discussions and critical comments on the manuscript. The work was supported by the Department of Energy’s Genomes to Life program, grant number DE-FG02-04ER63787.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Smith, H.E. The transcriptional response of Escherichia coli to recombinant protein insolubility. J Struct Funct Genomics 8, 27–35 (2007). https://doi.org/10.1007/s10969-007-9030-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10969-007-9030-7