Isolation of chimaeric forms of elongation factor EF-Tu by affinity chromatography

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Abstract

Six different recombinant chimaeric forms of a three-domain protein, proteosynthetic elongation factor Tu (EF-Tu), composed of domains of EF-Tu of mesophilic (Escherichia coli) and thermophilic (Bacillus stearothermophilus) origin as well as free N-terminal domains of EF-Tu, and the whole recombinant EF-Tus of both organisms were prepared and isolated by the GST (glutathione S-transferase) fusion technology. Several modifications in the standard isolation and purification procedures are described that proved necessary to obtain the proteins in a purified and undegraded form.

Introduction

Elongation factor Tu (EF-Tu) is a protein ubiquitous in all kingdoms. It plays a central role in protein biosynthesis, where it serves in the GTP-bound form for the transport of aminoacyl-tRNA to the A-site of the mRNA-programmed ribosome. The factor also possesses a low intrinsic GTPase activity, it is a GTPase (see Ref. [1] for a review). Elongation factors Tu form a family of proteins highly homologous in primary, secondary and tertiary structure. This may be very useful for the study of evolutionary relationships between all organisms as well as for the elucidation in protein molecules of structural features of adaptation to various living conditions.

The elements of thermostability in the molecule of elongation factor Tu (EF-Tu, Mw 43,290 Da [2]) of the moderately thermostable B. stearothermophilus (growth optimum 55–62°C) were investigated by creating recombinant mesophile/thermophile chimaeric forms of this three-domain protein. The chimaeric EF-Tus were composed of domains of EF-Tu from this organism combined with domains of the highly homologous (75% amino acid identity) but mesophilic EF-Tu (Mw 43,200 Da [3]) from E. coli (growth optimum 37°C). Although domain 1 (N-terminal or G-domain) of EF-Tu is the site of GDP/GTP binding and GTPase activity of the protein [4], [5], [6], the presence of all three domains is necessary for the binding of aminoacyl-tRNA and the function of the protein in protein biosynthesis [1]. To enable the separation of the recombinant proteins, overexpressed in E. coli, from the cellular E. coli EF-Tu, the most abundant protein in the cell, the recombinant proteins were fused with glutathione S-transferase (GST) and purified by affinity chromatography on Glutathione Sepharose 4B [7].

This approach has already proved useful for the preparation of many proteins and also for the preparation of EF-Tu from two organisms. E. coli GST–EF-Tu and its mutant forms [8], truncated forms of E. coli GST–EF-Tu [9] and a GST form of the wild type EF-Tu from Bacillus subtilis [10] were isolated by the column methods. We describe here several modifications of the standard batch isolation procedure (recommended by the manufacturer) that proved necessary to obtain 10 various recombinant forms of EF-Tu in good yield and pure and undegraded state.

Section snippets

Chemicals and reagents

Glutathione Sepharose 4B, pGEX-5X-3 expression vector, factor Xa protease, reduced glutathione and [3H]GDP (10 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Prague, Czech Republic). Triton X-100 and phosphoenolpyruvate were obtained from Sigma (Prague, Czech Republic). 2-Mercaptoethanol, phenylmethyl sulphonylfluoride (PMSF), GDP (Na-salt) and GTP (Na-salt) were from Serva (Prague, Czech Republic). Isopropyl β-d-thiogalactoside (IPTG) was purchased from Amersham Pharmacia Biotech or

Results and discussion

Modifications of the standard GST isolation procedure introduced mainly in the GS4B binding and washing steps and in the factor Xa cleavage step that are described in the Methods and below resulted in the preparation of highly purified and undegraded recombinant products (see Fig. 3 to compare the purity of EF-Tu proteins prepared by the standard and by the modified procedure). The following recombinant EF-Tu proteins were obtained by this modified method in a pure state: recombinant E. coli (Ec

Acknowledgements

The work was supported by the Grant Agency of the Czech Republic, grant no. 204/98/0863.

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