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

Abstract

The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37^oC, 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 (2^4-1) design at two levels: (i) the effect of storing (24-48 h) the seeded broth at 4oC prior to incubation at 37^oC; (ii) the effect of agitation speed (100-200 rpm); (iii) the final concentration (0.05-0.5 mM) of IPTG (isopropyl–β-D-thiogalactopyranoside); and (iv) the addition of IPTG at set cell densities (OD₆₆₀ 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^oC/24 h provided the highest yields of GFPuv under the conditions: (i) pre-storage at 4^oC/24 h; (ii) agitation speed at 100 rpm; (iii) 0.5 mM IPTG; (iv) IPTG addition at OD₆₆₀=~0.01. On the other hand, at 37^oC/ 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.


Key Words: Green fluorescent protein (GFPuv), Escherichia coli DH5-α, growth kinetic parameters, expressed GFPuv kinetic parameters, three phase partitioning extraction (TPP).


African Journal of Biotechnology Vol.3(1) 2004: 105-111