Inclusion bodies, expressing

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. 

Figure 4. Growth (o) and inclusion body expression ( ) for a typical culture. The inoculum density was 6 CFU/mL.

Figure 5. Influence of inoculum density on growth (open symbols) and inclusion body expression (closed symbols) in CY15070/pWHA43 (a) 140 CFU/mL (b) 14 CFU/mL (c) 1.4 CFU/mL (d) 0.6 CFU/mL.

Figure 8. Master growth and inclusion body expression curves for inocula of...

After this screening, it became clear that intracellular expression of IL-4 v forming inclusion bodies in E. coli was by far the most suitable and productive system. With many of the evaluated systems it was possible to express IL-4 v, but the yield was generally rather low as compared to the E. coli inclusion body expression system. Therefore, it was clear that this system should be optimized for the intracellular expression of IL-4 v inclusion bodies in E. coli. 

Table 12.1 Criteria for evaluation of an E. coli inclusion body expression system...

Fradkin AH, Boand CS, Eisenberg SP, Rosendahl MS, Randolph TW. Recomhinant murine growth hormone from E. coU inclusion bodies expression, high-pressure solubilization and refolding, and characterization of activity and structure. Biotechnol Prog 2010 26 743-749. 

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. 

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