Novel Modification of Growth Medium Enables Efficient E. coli Expression and Simple Purification of an Endotoxin-Free Recombinant Murine Hsp70 Protein

• Published in: Journal of microbiology and biotechnology Vol. 19; no. 7; pp. 727 - 733
• Main Authors: Zachova, Katerina, Palacky University in Olomouc, Olomouc, Czech Republic, Krupka, Michal, Palacky University in Olomouc, Olomouc, Czech Republic, Chamrad, Ivo, Palacky University in Olomouc, Olomouc, Czech Republic, Belakova, Jana, Palacky University in Olomouc, Olomouc, Czech Republic, Horynova, Milada, Palacky University in Olomouc, Olomouc, Czech Republic, Weigl, Evzen, Palacky University in Olomouc, Olomouc, Czech Republic, Sebela, Marek, Palacky University in Olomouc, Olomouc, Czech Republic, Raska, Milan, Palacky University in Olomouc, Olomouc, Czech Republic
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.07.2009; 한국미생물·생명공학회
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Affinity purification; Animals; Biological and medical sciences; Biotechnology; Chromatography, Affinity; Culture Media - chemistry; endotoxin removal; Endotoxins - biosynthesis; Endotoxins - isolation & purification; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; GlcNAc; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) - biosynthesis; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) - isolation & purification; HEAT SHOCK PROTEINS; hsp70; HSP70 Heat-Shock Proteins - biosynthesis; HSP70 Heat-Shock Proteins - isolation & purification; Industrial Microbiology - methods; MALDI-TOF; Mice; protein expression; PROTEINAS DE SHOCK TERMICO; PROTEINE DE CHOC THERMIQUE; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; 생물학; Purification; MALDI-TOF; Growth; Rodentia; Matrix assisted laser desorption ionization; Endotoxin; Affinity purification; Toxin; hsp70; Vertebrata; Mammalia; Mouse; GIcNAc; endotoxin removal; Affinity; Recombinant protein; protein expression
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.0810.570

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His-tagged proteins expressed in E. coli.