Purification of a gene product for characterization or antibody production is greatly simplified by cloning and expressing the gene in question, usually fused to an affinity tag, in Escherichia coli. However, the heterologous expression approach does not always allow formation of multiprotein complexes. In these cases, the complexes should be assembled in and isolated from the original host. A generalized method (tandem affinity purification) for protein complex purification from yeast has been described (2, 32). In this method, two tags are fused to the target protein of interest, and proteins interacting with the target are isolated by using two successive affinity purification steps. The components of protein complexes are later separated in and isolated from sodium dodecyl sulfate (SDS)-polyacrylamide gels for mass spectrometric (MS) identification. Tools that can facilitate a similar approach in a wide range of bacteria have not been developed yet.
Protein overproduction in E. coli sometimes has other limitations, especially when a foreign gene is expressed. No expression or a low efficiency of expression, degradation, toxicity, and protein insolubility are the most common problems. Providing other subunits and factors needed for posttranslational modification, such as processing of signal sequences, protein cleavage, folding, and incorporation of prosthetic groups, is also problematic, and the absence of these subunits and factors results in an inactive protein (27). Some of these problems can be solved if the protein is expressed and purified from the original bacterial host by employing specific expression vectors or one of the broad-host-range expression vectors available (4, 5, 13, 15). Usually, these are not available commercially, and it is hard to find one that fulfills all the requirements needed for a particular study or organism. Existing vectors are complicated to redesign; moreover, it is laborious and time-consuming to change or add required properties because of the lack of sequence data, the large size, and often the need for several cloning steps.
Our modular concept was to combine a broad-host-range vector backbone, containing all the necessary properties generally needed for protein expression and purification, with the possibility of easy insertion of desired promoters or replacement of various features. The resulting vectors are small and mobilizable, and their sequences are known. Different fusion tags are available to help protein purification, or they can be omitted if desired. The tandem FLAG-tag (17)-Strep-tag II (35) combination was designed to allow purification and study of protein complexes. Promoter regions from Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus, inserted upstream from the expression cassettes, were utilized to express proteins in these hosts at different levels depending on the inserted promoter's activity. In addition, it was demonstrated that the same construct was able to overproduce the protein in the appropriate E. coli host.
The tandem FLAG-tag-Strep-tag II combination was utilized in a study of hydrogenase maturation in T. roseopersicina. Assembly of the active site, located in the large subunit of hydrogenases (containing Ni, Fe, CO, and CN−), is a complex process assisted by several proteins (8, 11). Two of the hydrogenase maturation-assisting proteins of T. roseopersicina (HypC2 and HupK) (28) were used in coaffinity purification experiments to test the utility of the tandem FLAG-tag-Strep-tag II combination for detecting protein-protein interactions and its usefulness for studying hydrogenase maturation.