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

The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

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Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3732 ◽

2003 ◽

Vol 50 (1) ◽

pp. 239-247 ◽

Cited By ~ 3

Author(s):

Anna-Maria Ochocka ◽

Marzena Czyzewska ◽

Tadeusz Pawełczyk

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Rho Gtpase ◽

Expression System ◽

Cloning And Expression ◽

Soluble Form ◽

E Coli ◽

Escherichia Coli Cells ◽

Step Procedure ◽

Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

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