Biopolymers and cell. 2003; 19 (4): 367 - 373

Overproduction of human alpha-2b interferon in a soluble form in Escherichia coli cells using the bacteriophage T7 RNA polymerase-base expression system

I. Yu. Slavchenko, E. V. Boreyko, N. V. Vorobcy, S. I. Chernykh, V. A. Kordyum

An effective technology of soluble human alpha-2b interferon (IFN) production using the bacteriophage T7 polymerase-base expression system have been developed. For this purpose an artificial gene for human alpha-2b interferon (IFN) has been chemically synthesized and cloned into the expression vector pET-24a containing T7 promoter and terminator. A recombinant plasmid named pET-alFN was transformed into E. coli BL (DE3) cells and synthesis of the IFN was induced with 1PTG. The influence of temperature (37 and 21 °C) of E. coli BL (pET-alFN) cells cultivation on the level of synthesis and the yield of soluble IFN have been investigated. We found that level of recombinant protein synthesis at 37 °C is higher than at 21 °C in the first hours after induction. Thus, the IFN yield after 4 hour postinduction amounts about 20 % at 37 °C and only 7 % of the total cellular proteins at 21 °C. However, it has been determined that the expression of IFN at 37 °C results in accumulation of recombinant protein in cells as the insoluble inclusion bodies. It was shown that the lower temperature promotes accumulation of target protein in a soluble form and that soluble IFN can be produced in E. coli BL (pET-alFN) cells in higher amounts by postinduction cultivation at 21 °C during 18 h. The technology of IFN production developed by us includes the phage lambda infection of E. coli BL21 (DE3) cells, containing a plasmid with a target gene under the T7 phage promoter. The process results in lysis of the producer strain cells and releasing newly synthesized target protein into the growth medium, where it accumulates in a soluble form. Under the optimal cultivation conditions 1 liter of phage lysate contains about 200 mg of a soluble IFN. The obtained yield of soluble IFN is more than 2,5 times higher compared to the previously achieved yield of a soluble IFN using as a producer E. coli SO30 (pIF-16) carrying the plasmid with the two copies of an artificial gene for IFN under the control of the Ptrp tandem promoter.