Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

Abstract

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

“…The medium pH was adjusted at 5, 6, 7 or 8 and its effects on soluble expression of GST-hD2 were investigated. 28 To examine heat and osmotic shock responses, the study were performed according to the protocol by Oganesyan et al 12 with some minor modification. Harvesting of cells were performed 3 and 15 h after induction.…”

“…[6][7][8] There are several approaches to avoid the production of inclusion body in bacteria such as optimization of the cultivation temperature, type and the concentration of inducers, medium additives, and utilization of heat and osmotic shocks. [9][10][11][12][13] Escherichia coli is one of the common host prokaryotic organisms for the production of recombinant proteins because of facile genetic engineering and optimization of recombinant protein expression. 14,15 Total amount of recombinant protein has an important role in its degradation rate.…”

“…The presence of some additives like sorbitol and sodium chloride in the medium triggers osmotic shock, and the high incubation temperature for a short period of time produces a heat shock response. 12 Either osmotic or heat shocks cause increased manufacturing of chaperone molecules which play essential roles in proper folding of de novo synthesized proteins in cells at harsh conditions and eventually catalyze proper folding of proteins. 12,18 Metal ions have described as solubilizing factors for some recombinant proteins.…”

“…12 Either osmotic or heat shocks cause increased manufacturing of chaperone molecules which play essential roles in proper folding of de novo synthesized proteins in cells at harsh conditions and eventually catalyze proper folding of proteins. 12,18 Metal ions have described as solubilizing factors for some recombinant proteins. 20 Especially magnesium solubilization activity is proposed to be higher than any other metal ions.…”

Structural Genomics of Minimal Organisms: Pipeline and Results

“…We have tested a protocol that combines heat shock and high salt growth (Oganesyan et al, 2006). The cells were grown in the presence of 0.5 M NaCl and incubated at 47 o C at the beginning of induction with IPTG for 20 minutes.…”