The effects of protein solubility on the RNA Integrity Number (RIN) for recombinant Escherichia coli

Abstract

High quality, intact messenger RNA (mRNA) is required for DNA microarray and reverse transcriptase polymerase chain reaction analysis and is generally obtained from total RNA isolations. The most widely recognized measure of RNA integrity is the RNA Integrity Number (RIN) obtained from the Agilent Bioanalyzer, as it provides sizing, quantification, and quality control measures. This work describes comparisons of the RIN values obtained for recombinant E. coli. Uninduced recombinant E. coli cultures were examined, as well as induced cultures that produced either a soluble or insoluble recombinant protein. The uninduced cultures and the induced cultures producing soluble protein had higher RIN values than the induced cultures producing insoluble protein. These lower RIN values for E. coli producing the insoluble protein indicate that cellular degradation of the ribosomal RNA species is the likely cause of the lower RIN values. As the use of DNA microarrays and other gene expression tools increase in usage in the industrial recombinant protein production community, these results suggest the need for further studies to determine acceptable RIN ranges for gene expression analysis and effects of various culture conditions on RIN values for recombinant E. coli.

Dynamic transcriptional response of Escherichia coli to inclusion body formation

“…The overall objective of this study was to characterize the dynamic gene expression variability in E. coli due to insoluble and soluble protein production. The pTVP1GFP and pGFPCAT plasmids and the VP1GFP and GFPCAT proteins were selected to minimize differences between the culture conditions, except for protein solubility (Salazar et al, ). E. coli MG1655 pTVP1GFP and pGFPCAT were cultured in synchronized shake flasks to produce either the mostly insoluble VP1GFP or the soluble GFPCAT proteins, respectively.…”

“…The plasmid pTVP1GFP (gift from A. Villaverde) encodes the VP1 capsid of foot‐and‐mouth disease (Liu et al, ) fused to green fluorescent protein (GFP) (Garcia‐Fruitos et al, ). The pGFPCAT plasmid was constructed from the pTrcHis‐GFP UV /CAT plasmid (gift from WE Bentley) (Cha et al, ), where the GFP UV was replaced with the GFP from the pTVP1GFP plasmid; however, the GFP protein is located on the amino‐terminus of the VP1 protein and on the carboxyl‐terminus of the CAT protein (Salazar et al, ). E. coli MG1655 were transformed with either the pTVP1GFP or pGFPCAT plasmid.…”