Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus

Abstract

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

FIG. 1.

Schematic representation of S. solfataricus entry vectors. The diagrams show the regions of the pBR322 vector with the pyrEF gene cassette, the different promoters, and the restriction sites that can be used to clone the gene of interest behind the promoter sequences.

FIG. 2.

β-Galactosidase activity monitored in a nondenaturing polyacrylamide gel before and after induction with arabinose. Crude extracts (50 μg of protein) of two independent pSVA9 transformants were separated by blue PAGE 0, 22, and 46 h after the addition of 0.4% arabinose to the growth medium. (Left) ONPG activity staining of β-galactosidase. (Right) Colloidal Coomassie blue-stained gel.

FIG. 3.

Purification and activity of A. ambivalens Sor isolated from S. solfataricus transformed with pMJ05-sor. (A) SDS-PAGE of different fractions (E1 and E2) eluted from the Streptactin column. M, molecular mass marker proteins. Solid arrow, Sor band; dashed arrow, AccC subunit of the acetyl/propionyl-CoA carboxylase (both identified by MALDI-TOF analysis). (B) Sulfur oxygenase and reductase activities of pooled eluate fractions showing time-dependent increases in the amounts of thiosulfate and hydrogen sulfide in the assay mixture (19). Background, nonenzymatic production of thiosulfate and hydrogen sulfide from sulfur disproportionation under the same assay conditions without the enzyme (19).

FIG. 4.

Western analysis of FlaI expression. S. solfataricus cells transformed with pSVA6 and control PH1-16 cells were grown for 2 days at 88°C. Samples were taken at the beginning of the temperature shift from 78 to 88°C and after 1 and 2 days of growth at 88°C. Membranes were isolated from the cells and were analyzed by SDS-PAGE and immunoblotting using FlaI-specific antibodies. The arrow indicates the detected expression product.

FIG. 5.

Overexpression, purification, and activity of SSO2680. (A) Coomassie blue-stained SDS-PAGE gel of His tag-specific affinity chromatography fractions from solubilized membranes derived from S. solfataricus cells transformed with pSVA15. In the lower panel, the corresponding Western blot of the same samples, using His tag-specific antibodies, is shown. M, molecular mass marker; St, starting material; FT, flowthrough; W, wash; E, elution fraction. (B) ATPase activity of purified SSO2680 in the presence of EDTA, Mg²⁺, or Mn²⁺. (C) Overexpression of SSO2680. Membranes of a single pSVA15 transformant were separated in a Coomassie blue-stained SDS-PAGE gel before (−) and after (+) induction with 0.4% arabinose. The arrow indicates SSO2680. In the lower panel, the corresponding Western blot of the same samples, using His tag-specific antibodies, is shown.

FIG. 6.

Purification and gel filtration of SSO0287. (A) Coomassie blue-stained SDS-PAGE gel of Strep tag-specific affinity chromatography fractions of the cytoplasm of SSO0287 transformants. St, starting material; FT, flowthrough; E, elution fraction. (B) Gel filtration of purified SSO0287. Traces were recorded at 280 nm (black line) and 410 nm (dashed line).