Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green florescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar K M values, but SUMO protease had a 25-fold higher k cat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein and 5-lipoxygenase-activating protein (FLAP) are among a large number of membrane proteins that are poorly expressed when traditional expression systems and methods are employed. Therefore to efficiently express difficult membrane proteins, molecular biologists will have to develop novel or innovative expression systems. To this end, we have expressed the SARS-CoV M and FLAP proteins in Escherichia coli by utilizing a novel gene fusion expression system that takes advantage of the natural chaperoning properties of the SUMO (small ubiquitin-related modifier) tag. These chaperoning properties facilitate proper protein folding, which enhances the solubility and biological activity of the purified protein. In addition to these advantages, we found that SUMO Protease 1, can cleave the SUMO fusion high specificity to generate native protein. Herein, we demonstrate that the expression of FLAP and SARS-CoV membrane proteins are greatly enhanced by SUMO fusions in E. coli.

Natural anti-proteases (alpha 1-protease inhibitor (alpha 1-PI; alpha 1-antitrypsin) and alpha 2-macroglobulin (alpha 2-M)) were found in the blood of rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis. The alpha 2-M inhibited Cryptobia salmositica proteases and was significantly higher in brook charr than in rainbow trout. Under in vitro conditions it took longer for the same number of parasites to neutralize the alpha 2-M in charr than in trout blood. The haemolysis which occurred when C. salmositica was incubated in the blood of rainbow trout was due to neutralization of alpha 2-M. This in vitro study also showed that it was the metalloprotease of C. salmositica that lysed red blood cells and the plasma of the two species of fishes initially prevented haemolysis by inhibiting the proteolytic activity. We suggest that the natural plasma alpha 2-M plays an important role in defence against cryptobiosis in fishes.

Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.

Proteases were detected in Cryptobia salmositica (pathogenic and nonpathogenic vaccine strains), C. bullocki, and C. catostorni using azocasein and hide powder azure as substrates. Maximum activity occurred in acidic pH and the pathogenic strain of C, salmositica had the highest activity.Cysteine protease was found in pathogenic and nonpathogenic Cryptobia spp., but metallo-protease was only present in the pathogenic strain of C. salmositica. Five enzyrnatic bands were detected in the pathogenic C. salmositica using haemoglobin-SDS-PAGE: four of these were cysteine proteases (49,60, 66 and 97 kDa) and the other was a metallo-protease (200 kDa). The pathogenic C. salmositica lost the metallo-protease after 10 mo of in vitro culture. We suggest that the metallo-protease of the pathogenic C. salmositica is related to pathogenicity of the parasite.