Expression inclusion body

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

P. fluorescens has well-developed mechanisms for the secretion of proteins into the periplasm which facilitates S—S bond formation and proper N-terminal processing. It also allows one to reduce the formation of inclusion bodies and, thus, the additional costs caused by refolding processes. Proteolytic degradation of the expressed protein is also low, and very high... 

Ralstonia eutropha, a Gram-negative bacterium that has the special feature of high-level expression without inclusion body formation, can also be regarded as a competitor to E. coli [59]. 

Occasionally, however, driving the overexpressed protein into inclusion bodies can be advantageous, for example if the protein is unfolded and protease-sensitive. Forcing proteins into inclusion bodies can be achieved by increasing the expression temperature (to approximately 40 °C) and by using more IPTG. Alternatively, special bacterial overexpression plasmids exist that target a protein into inclusion bodies. 

High-level expression of full-length proteins in E. coli may result in the production of inclusion bodies. These are insoluble, inactive aggregates resulting from inappropriate folding and association via hydrophobic interactions. The proteins are fimction-ally inactive in their aggregated state. The formation of inclusions can be advantageous for purification, provided the protein can be successfully solubilized and renatured into its active form. This involves isolation of inclusions and removal of unwanted... 

Upadhyay, P., Patra, A. K., Mukhopadhyay, R., and Panda, A. K. (2001). Real time detection and quantification of inclusion bodies expressed in Escherichia coli by impedance measurements. Biotechnol. Lett. 23, 839-843. 

La Vallie, E. et al. (1993). A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio j Technology 11, 187-193. 

ImHNL is a nonglycosylated homodimer (84 kDa) which catalyzes the reversible cleavage of aliphatic (R)-cyanohydrins [34]. This HNL does not require complex protein modification after protein biosynthesis. Thus, expression in prokaryotic Escherichia coli) and eukaryotic hosts Pichia pastoris) is possible [35-37]. However, initial trials to express IwHNL in E. coli were hampered by formation of inclusion bodies [36]. 

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