Characterization of protein expression in recombinant systems has been enhanced through the availability of techniques such as Western blot analysis and enzyme-linked immunosorbent assay (ELISA) (1). A limitation of these techniques is the need for an antibody specific to the protein of interest. The development of epitope tags such as c-myc and HA has addressed this limitation by providing the means to characterize recombinant proteins without the need for a protein-specific antibody (2,3). While they provide good utility, techniques such as Western blotting involve multiple incubation and wash steps that require a significant amount of time to perform. Thus, there exists a need for a more efficient method to detect and characterize recombinant proteins. We have developed an alternative blot assay, which provides similar utility to Western blotting without the need for specific antibodies or multiple incubation and wash steps ((Table 1)).

The ability to detect recombinant proteins using this novel blotting technique takes advantage of enzyme fragment complementation (EFC), whereby a short peptide (the α fragment or enzyme donor) derived from the N terminus of β-galacto-sidase (β-gal) can reconstitute enzyme activity in a corresponding deletion mutant of full-length β-gal (the enzyme acceptor or EA) (4). Variants of the α fragment optimized for recombinant expression and high complemented enzyme activity have been created and are known as ProLabel™ (PL; S SNSLAVVLQRRDWENPGVTQLN RLAAHPPFASWRNSEEARTDRPS QQLRSLNGE; DiscoveRx, Fremont, CA, USA) (5). Since EA essentially lacks β-gal activity in the absence of PL, EFC technology is ideally suited for use in homogenous assays that can specifically and quantitatively measure PL availability for complementation (5,6,7). The novel blot assay extends this technology to the detection of recombinant proteins on a solid support, such as a nitrocellulose or polyvinylidine difloride (PDVF) membrane.

To demonstrate the utility of the EFC blot assay, we examined its ability to detect recombinant proteins in extracts prepared from CHO cells transfected with a variety of PL-tagged constructs. These examples include a stably transfected cell line expressing the nuclear factor of activated T cells protein tagged at its C terminus with ProLabel (NFAT-PL) (8,9) as well as transiently transfected cells expressing c-Jun-PL and cyclin D-PL fusion proteins. Proteins extracted from cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane using standard techniques. An additional fusion protein consisting of NeutrAvidin™ chemically conjugated to PL (NA-PL) was included as a control. The blots were incubated for 1 h with EA reagent (DiscoveRx) to enable complementation with the membrane-bound PL. Chemiluminescent β-gal substrate (DiscoveRx) was then added directly to the EA solution in which the blot was immersed. Since noncomplemented EA is inactive, it is not necessary to remove or wash excess EA from the membrane. After a 15-min incubation, the blots were drained of excess liquid, placed between sheets of clear plastic, and the β-gal chemiluminescent activity was detected using a digital imaging system.

A 5-min exposure produced sufficient signal to detect NFAT-PL in a cell lysate sample equivalent to 4 µg total protein ((Figure 1)). A major band with an apparent molecular weight of 85 kDa was detected, which is consistent with the expected mass of the NFAT-PL fusion protein (86.5 kDa). Two minor bands were also detected: one of approximately 60 kDa is consistent with it being a proteolytic cleavage product of full-length NFAT-PL, and the other of approximately 6 kDa is consistent with it being the PL peptide moiety (55 amino acids, approximately 6.3 kDa) liberated by proteolytic cleavage. Transiently expressed c-Jun-PL and cyclin D-PL of approximately 45 and 42 kDa were detected in cell lysates equivalent to 0.5 and 0.35 µg total protein, respectively. Control cell extracts from CHO cells transiently transfected with vector only lacked all bands. The NA-PL control sample exhibited three major bands: the 22 and 27 kDa bands are consistent with the expected molecular weights of a neutra-vidin monomer conjugated with one or two PL peptides, respectively, and the 5-kDa band is consistent with the free unconjugated PL peptide used in the synthesis (47 amino acids, approximately 5.5 kDa). There is likely to be little limitation in the size of potential fusions detected by EFC blot, since low molecular weight PL is readily detected in both the NA-PL control and in the inferred cleavage products of other full-length recombinant proteins.