Biomaterial Database

Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing Heat-shock proteins. 1996

J G Thomas, and F Baneyx

Department of Chemical Engineering, University of Washington, Seattle 98195-1750, USA.

PreS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6 fold increase in the recovery of enzymatically active preS2-S'-beta-galactosidase over a wide range of growth temperatures (30-42 degrees C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30 degrees C only. Co-immunoprecipitation experiments confirmed that preS2-S'-beta-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42 degrees C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor sigma 32, or by addition of 3% ethanol (v/v) to the growth medium, a 2-3 fold higher recovery of active enzyme was observed at 30 and 42 degrees C, but not at 37 degrees C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S'-beta-galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D011993	Recombinant Fusion Proteins	Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.	Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D002479	Inclusion Bodies	A generic term for any circumscribed mass of foreign (e.g., lead or viruses) or metabolically inactive materials (e.g., ceroid or MALLORY BODIES), within the cytoplasm or nucleus of a cell. Inclusion bodies are in cells infected with certain filtrable viruses, observed especially in nerve, epithelial, or endothelial cells. (Stedman, 25th ed)	Cellular Inclusions,Cytoplasmic Inclusions,Bodies, Inclusion,Body, Inclusion,Cellular Inclusion,Cytoplasmic Inclusion,Inclusion Body,Inclusion, Cellular,Inclusion, Cytoplasmic,Inclusions, Cellular,Inclusions, Cytoplasmic
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006360	Heat-Shock Proteins	Proteins which are synthesized in eukaryotic organisms and bacteria in response to hyperthermia and other environmental stresses. They increase thermal tolerance and perform functions essential to cell survival under these conditions.	Stress Protein,Stress Proteins,Heat-Shock Protein,Heat Shock Protein,Heat Shock Proteins,Protein, Stress
D001616	beta-Galactosidase	A group of enzymes that catalyzes the hydrolysis of terminal, non-reducing beta-D-galactose residues in beta-galactosides. Deficiency of beta-Galactosidase A1 may cause GANGLIOSIDOSIS, GM1.	Lactases,Dairyaid,Lactaid,Lactogest,Lactrase,beta-D-Galactosidase,beta-Galactosidase A1,beta-Galactosidase A2,beta-Galactosidase A3,beta-Galactosidases,lac Z Protein,Protein, lac Z,beta D Galactosidase,beta Galactosidase,beta Galactosidase A1,beta Galactosidase A2,beta Galactosidase A3,beta Galactosidases
D013696	Temperature	The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.	Temperatures
D013997	Time Factors	Elements of limited time intervals, contributing to particular results or situations.	Time Series,Factor, Time,Time Factor
D017510	Protein Folding	Processes involved in the formation of TERTIARY PROTEIN STRUCTURE.	Protein Folding, Globular,Folding, Globular Protein,Folding, Protein,Foldings, Globular Protein,Foldings, Protein,Globular Protein Folding,Globular Protein Foldings,Protein Foldings,Protein Foldings, Globular
D050956	HSP40 Heat-Shock Proteins	A family of heat-shock proteins that contain a 70 amino-acid consensus sequence known as the J domain. The J domain of HSP40 heat shock proteins interacts with HSP70 HEAT-SHOCK PROTEINS. HSP40 heat-shock proteins play a role in regulating the ADENOSINE TRIPHOSPHATASES activity of HSP70 heat-shock proteins.	DnaJ Protein,HSP40 Heat-Shock Protein,HSP40 Protein,Heat-Shock Proteins 40,DnaJ Proteins,HSP40 Protein Family,HSP40 Proteins,Heat Shock Protein 40 Family,Heat-Shock Protein 40,HSP40 Heat Shock Protein,HSP40 Heat Shock Proteins,Heat Shock Protein 40,Heat Shock Proteins 40,Heat-Shock Protein, HSP40,Heat-Shock Proteins, HSP40,Protein, DnaJ,Protein, HSP40,Protein, HSP40 Heat-Shock,Proteins, HSP40 Heat-Shock