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Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

ABSTRACT: The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low ?-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

SUBMITTER: Upadhyay AK

PROVIDER: S-EPMC3315509 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

Upadhyay Arun K AK Murmu Aruna A Singh Anupam A Panda Amulya K AK

PloS one 20120329 3

The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, t ...[more]

PMID: 22479486

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Project description:The ability to control the synthesis of products in the absence of cell division remains an attractive alternative for the optimization of microbial processes. If this could be achieved, it would dramatically expand the productivity of many microbial processes by increasing product synthesis in the absence of an increase in cell mass. In the present work we have looked at the factors involved in the design of a host where the fluxes would be channeled towards product formation rather than biomass synthesis. To identify the genes responsible for diverting the metabolic flux specifically towards product formation we have used for this study is quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabolically active cells. These cells channel the metabolic flux towards recombinant protein production and therefore the specific product yield per unit biomass is significantly higher. Indole which is previously known to be a signalling molecule is here used as an inducer of Quiescence. E.coli L-Asparaginase is used as a model protein in this work. Fed batch cultures were carried out in Sartorius BIOSTAT B plus 5liter bioreactor. The data acquisition was done with the MFCS Shell software (version) provided with the bioreactor. The Sartorius BIOSTAT B plus had proportional integral derivative-based control loops for temperature, pH, antifoam, and dissolved-oxygen (DO) regulation. The culture was grown in a batch mode till 10-12 OD (µ=0.5) and then the feed was attached. In the next hour the culture was induced for product formation with 1M IPTG. In case of Q culture quiescence was induced with 0.5M Indole just 5min after induction with IPTG. Post induction every hour samples were collected and frozen with liquid nitrogen and stored at -80ºC for further analytical use. Total RNA extraction, RNA quality control (Agilent BioAnalyser), total RNA labeling, microarray hybridization and scanning were performed according to the Affymetrix GeneChip Expression analysis .

Dataset Information

Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

Publications

Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

Similar Datasets

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