Optimization of recombinant β-NGF expression in Escherichia coli using response surface methodology.
Human nerve growth factor a member of the neurotrophin family can be used to treat neurodegenerative diseases. As it has disulfide bonds in its structure, periplasmic expression of it using appropriate signal sequence is beneficial. Therefore, in this work β-nerve growth factor (β-NGF) was expressed in Escherichia coli using pET39b expression vector containing DsbA signal sequence. In an initial step, the effect of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose concentration as inducer on protein production was investigated using response surface methodology. Then the effect of different postinduction time and temperature on protein production was studied. Our results indicated that the highest β-NGF production was achieved with 1 mM IPTG and low concentrations of lactose (0-2% w/v), low cultivation temperature of 25°C and postinduction time of 2 hr. Also following β-NGF purification, bioassay test using PC12 cell line was done. The biological activity of the purified β-NGF showed a similar cell proliferation activity with the standard recombinant human β-NGF. In conclusion, the results indicated an optimized upstream process to obtain high yields of biologically active β-NGF.
a Department of Life Science Engineering, Faculty of New Sciences and Technologies , University of Tehran , Tehran , Iran.|b Department of Microbial Biotechnology, School of Biology, College of Science , University of Tehran , Tehran , Iran.
Pouria
P
Gholami Tilko
a Department of Life Science Engineering, Faculty of New Sciences and Technologies , University of Tehran , Tehran , Iran.
Zahra
Z
Hajihassan
b Department of Microbial Biotechnology, School of Biology, College of Science , University of Tehran , Tehran , Iran.
Hamid
H
Moghimi
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007218:Industrial Microbiology / Q000379:methods; D007785:Lactose / Q000378:metabolism; D020932:Nerve Growth Factor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D020932:Nerve Growth Factor; D007785:Lactose
E. coli; response surface methodology; β-NGF expression