High throughput expression of proteins is often hampered by the failure of certain proteins to express in the particular E. coli host strain used for the study. The identification of a host strain compatible for a wide variety of proteins is desirable. In this study, the recombinant expression of therapeutic proteins Erythropoietin (EPO), Streptokinase (SK) and Tumor Necrosis Factor Receptor Extra cellular domain (TNFR ED) that vary widely in their chemical nature was studied in four different strains of E. coli namely BL21 (DE3), BL21 (DE3) pLys S, BL21 (DE3) Rosetta pLys S and GJ1158. Since there are no previous report for the analysis of expression and solubility of the above mentioned proteins we studied the same in various E. coli stains. Here we report that E. coli strain GJ1158 which uses salt induction was found to be the most suitable for overexpression of all the three proteins. Interestingly rare codons were found not to play any significant role in the expression. Protein toxicity and aggregation propensity were also studied. One of the major factors influencing expression was the tendency of the protein to aggregate which in turn influences folding and toxicity levels. The solubility of the proteins was inversely proportional to aggregation. Expression levels were in the order of TNFR ED < EPO < SK. In conclusion, it was observed that E. coli GJ1158, a strain known to decrease aggregation of proteins was found to be more suited for expression. This is the first time GJ1158 has been included in this kind of analysis for comparison of protein expression in various E. coli hosts.
Aim: To investigate the regulatory potential of the Nnat second intron within the Nnat/Blcap micro-imprinted domain. Materials & methods: Mice with deletion of Nnat second intron at the endogenous Nnat/Blcap micro-imprinted domain were used to examine the effect of Nnat second intron on the transcriptional regulation of the Nnat and Blcap genes. Results & conclusion: Deletion of Nnat second intron affected Nnat expression in cis leading to the loss of Nnat expression from the active paternal allele. Nnat second intron was found to have the characteristics of an imprint control region including allele-specific DNA methylation and histone modifications and it also regulated the epigenetic profile of Nnat promoter by acting as an enhancer. Nnat second intron was also found to be regulating the expression of the Blcap transcripts.
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