Proprietary Expression Technology

VLI Research Inc. has developed the ubiquitin-like protein-fusion (UBL) system to overcome the problems faced by traditional gene-fusion technologies. This novel fusion system dramatically enhances the expression and stability of under-expressed or non-expressed proteins and peptides in prokaryotes and eukaryotes, and promotes solubility and proper folding of insoluble proteins. In addition, the tags facilitate rapid protein purification.

The fusion system enables expression of a wide range of proteins. After extraction of crude UBL-fusion from cells UBL is removed with UBL protease – a robust and highly specific enzyme that rapidly cleaves UBL-fusion before the +1 N-terminus position of the fused protein. This cleavage is complete for all amino acids in the +1 position except Pro, thus enabling development of efficient and inexpensive processes for production of various proteins and peptides with N-termini other than Met. This feature avoids additional expensive and yield-reducing steps of enzymatic or chemical removal of N-terminal residue(s) to obtain a desired +1 position.

The problems of protein expression and purification are encapsulated in the following table. The VLI UBL fusion technology has solutions to most of these problems.

Ubiquitin-like proteins are small (~100 residues), heat-stable proteins with a highly compact globular structure. Most of them are very well expressed in E.coli and, when fused with other proteins, improve protein production, solubility and biological activity. The system developed by VLI Research Inc. offers a set of E.coli, yeast and baculovirus vectors that allow simple unidirectional cloning of PCR-amplified genes immediately after 6xHis-SUMO. The cloning design precludes the introduction of undesirable amino-acid residues at the junction. Following initial capture, the 6xHis-UBL-portion can be cleaved with UBL-specific protease, also supplied by VLI.

A simple expression cloning strategy for E.coli is described in the following diagram. The fusion protein is subsequently cleaved with the UBL specific hydrolase.

The UBL fusion dramatically enhances the level of expression of several proteins in E.coli and eukaryotic cells. The following example shows that expression of green fluorescence protein (GFP) is dramatically enhanced by fusion with UBL. The fusion protein remains soluble in when expressed at 37?C. In addition, the UBL-GFP is correctly folded, as indicated by its ability to fluoresce at the appropriate wavelength.

Another remarkable property of the UBL system is the cleavage properties of the UBL proteases. The proteases can cleave any junction between the UBL and the fusion protein. The only junction that is resistant to cleavage is that in which proline is present as the N-terminus of the fused protein. The properties of UBL protease are demonstrated by the cleavage of 20 UBL-GFPs in SDS-PAGE, shown below. The 20 UBL-GFPs analyzed in this experiment differ only in the identity of the amino acid that replaces the first residue of GFP (at the N-terminus).

A triple labeled NMR structural project was made successful by UBL fusion. A GST fusion of serine/threonine kinase was not expressed in soluble form in E.coli. VLI scientists designed a UBL-kinase fusion, and the protein was triple labeled with 13C, 15N and 2H. One hundred (100) mg of the kinase was expressed in soluble form. The protein was active in solution, and the structure of the protein was elucidated. The following SDS-PAGE analysis demonstrates the strategy of purification of triple labeled kinase from E.coli.

Another example of VLI’s system is highlighted by successful expression and purification of matrix metalloprotease (MMP). The catalytic domain of the protein has been expressed by other expression systems in E.coli, but the entire protein fraction is insoluble. Fusion of the MMP with UBL allowed expression of soluble protein in E.coli. SDS-PAGE analysis shows that the unfused MMP 13 was not expressed well in E.coli. Fusion with UBL dramatically enhanced the expression level and, most important, the majority of the protein was soluble.

MMP 13 protein was purified from soluble fractions of E.coli (lane 1). The fusion protein was cleaved from UBL by UBL protease (lane 2 and 3). The cleaved protein was further separated from the UBL by subtraction using a nickel column (lane 4). This example demonstrates that UBL fusion not only enhances protein expression, but also promotes solubility and correct folding of the protein. Almost 98% of the MMP protein was found to be catalytically active following purification and cleavage.