Expression of green fluorescent protein (GFPuv) in Escherichia coli DH5-a, under different growth conditions

• Published in: African journal of biotechnology 2004-01, Vol.3 (1), p.105-111
• Main Authors: Thereza, Christina Vessoni Penna, Marina, Ishii, Luciana.Cambricoli, de Souza, Olivia, Cholewa
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 1684-5315; 1684-5315
• DOI: 10.5897/AJB2004.000-2019

The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5- alpha grown in LB/amp broth at 37 degree C, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by a fractional factorial (2 super(4-1)) design at two levels: (i) the effect of storing (24-48 h) the seeded broth at 4 degree C prior to incubation at 37 degree C; (ii) the effect of agitation speed (100-200 rpm); (iii) the final concentration (0.05-0.5 mM) of IPTG (isopropyl- beta -D-thiogalactopyranoside); and (iv) the addition of IPTG at set cell densities (OD sub(660) 0.01-0.8). GFPuv was extracted from cells by the three phase partitioning method (TPP) and further purified with a methyl HIC column. The cultures grown at 37 degree C/24 h provided the highest yields of GFPuv under the conditions: (i) pre-storage at 4 degree C/24 h; (ii) agitation speed at 100 rpm; (iii) 0.5 mM IPTG; (iv) IPTG addition at OD sub(660)= similar to 0.01. On the other hand, at 37 degree C/8 h, GFPuv expression was dependent upon agitation of broth cultures at 200 rpm and the IPTG addition at the beginning of the growth exponential phase.