Figure 1. Expression tags are the key component of the fusion protein approach. Three different expression tags are indicated as A (oval), B (oval) or C (circle). They can be fused to either N- or C-termini of native target proteins. Since different expression tags render different functions, dual- or multitags can be fused to a single target protein to gain more than one function.

Figure 2. Green fluorescence protein (GFP) is used as a report tag for proper folding of fusion proteins. GFP is fused to the C-termini of a native target protein. When the target proteins are properly folded (i.e. become soluble or inserted into lipid bilayer), GFP also folds properly and emits bright green fluorescence under ultraviolet (UV) light. Visualization of GFP-tagged fusion proteins was carried out by taking fluorescent images of the living E. coli cells by a fluorescent microscope.

Figure 3. Schematic representation of trans and cis cleavage of fusion proteins. The nucleotide sequence encoding the amino acid sequence of TEVP cleavage site is shown here. (a) The trans method. For in vitro cleavage, purified TEVP is mixed with the purified fusion proteins at a molar ratio of approximately 1/50–1/200. Trans cleavage can also be carried out in vitro by expressing the fusion protein and TEVP as two separate polypeptides in the same cell. (b) The cis TEVP method. A fusion protein contains an expression tag, TEVP, TEVP cleavage site, and target protein expressed in a host cell. In this case, intracellular self processing of fusion protein occurs and yields a native target protein.

Figure 4. Schematic representation of four different strategies for parallel cloning fusion proteinexpression vectors. (a) TA-TOPO cloning. Tag polymerase has nontemplate-dependent TA that adds a single deoxyadenosine (A) to the 3¢ ends of PCR products. The linearized T vector has single, overhanging 3¢ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. (b) Sticky-end PCR cloning method for cloning a His6-SUMO dual fusion protein. Two separate PCR reactions were carried out using one forward primer and two reverse primers. The two PCR products were mixed and were 5¢-end phosphorylated by T4 polynucleotide kinase. After denaturing at 95°C and annealing at 65°C, ~50% of the products carried 5¢ blunt end and 3¢ EcoRI ends, and were ready for ligation into vectors. The vector shown here was engineered to carry an Sfo I site spanning the end of SUMO. The final expression plasmid can be used to produce recombinant His6-SUMO fused target protein, and such a recombinant protein can be cleaved Ulp1 (SUMO protease) and release the native target protein with methionine at its NH₂-end next to the Gly Gly end of SUMO. (c) Ligation-independent cloning (LIC) method. The PCR product of target gene was digested by T4 DNA polymerase (3¢ to 5¢ exonuclease) in the presence of dATP to generate single-strand overhanging. The single-stranded overhang allowed the target gene DNA to anneal with the LIC vector with complementary overhangs. The annealed product without ligation reaction was transformed directly into E. coli, in which covalent bonds formed at the vector-insert junctions to yield circular plasmids. This expression vector was designed to produce fusion proteins with an enterokinase cleavage site. After enterokinase cleavage, the fusion protein yielded a protein with native N-terminal amino acid sequence. (d) Recombinatorial cloning (RC). Donor vector with attL recombined with a destination vector with attR to form a new expression clone with attB and a byproduct with attP.