Native chemical ligation is a useful synthetic method to join independently generated protein fragments via a native peptide bond. Expressed protein ligation (EPL) is a form of native chemical ligation that utilizes intein technology for expression and/or purification of one or more of the fragments to be ligated [1
]. EPL has been used to incorporate unnatural amino acids [2
], biophysical probes [4
], post-translational modifications [5
] and isotope labels [6
] in specific locations within a ligated protein product [reviewed in 1
]. These and other EPL strategies have allowed unique problems of protein structure, folding, enzyme mechanism, ion channel function and signaling to be addressed in novel and insightful ways.
EPL involves joining the desired protein fragments via an autocatalytic chemical ligation [8
]. This reaction, which creates a peptide bond between protein fragments, requires a specific thioester linked leaving group moiety covalently bound to the terminal carboxyl group of one fragment and a cysteine at the amino terminus of the second fragment [8
]. Adaptations of intein-dependent protein splicing reactions (analogous to intron/exon splicing) originally observed in Saccharomyces cerevisae
have made it possible to isolate appropriately modified fragments for subsequent ligation [10
]. Recombinant production of the protein fragment containing a thioester-linked leaving group moiety normally includes thiol-dependent autocatalytic, intein-mediated cleavage of an engineered fusion protein. Generation of the second protein fragment has relied on three primary approaches including synthetic production by solid-phase peptide synthesis, proteolysis of recombinant proteins by in vitro
or in vivo
], or thiol and temperature dependent intein mediated fusion protein cleavage [15
; reviewed in 9
]. Given limitations on fragment length using solid phase peptide synthesis, the specificity and cost of in vitro
protease cleavage and production/yield issues with intein-mediated fusion protein cleavage, the EPL strategy employed requires careful consideration.
Signal peptidases located in the bacterial periplasmic space have been extensively utilized for high-yield production of recombinant proteins [18
]. The predictable and precise nature of leader peptidase cleavage at the pelB-protein junction combined with high protease activity with a cysteine at position −1 [18
], suggests EPL-active protein fragments can be generated by engineering a pelB leader sequence adjacent to the amino-terminal cysteine of the fragment of interest. Furthermore, the pelB sequence directs the newly synthesized protein to the periplasmic space where the membrane-anchored peptidase is localized [20
]. Studies of bacterially expressed recombinant apolipoproteins have shown that, not only does efficient pelB cleavage occur, the protein product also escapes the periplasm and accumulates in the extracellular culture media [22
]. This process, for which the mechanism is unknown, facilitates recovery and downstream processing of recombinant proteins from bacterial cultures.
Apolipoprotein E (apoE) is a 299-amino acid glycoprotein that is a well-characterized ligand for the low-density lipoprotein receptor [26
]. The X-ray crystal structure of the isolated N-terminal domain revealed a globular bundle of four elongated amphipathic α-helices that is stabilized by interhelical hydrophobic interactions in the absence of lipid [27
]. Likewise, insect apolipophorin III (apoLp-III) adopts a helix bundle organization [28
]. Using recombinant DNA technology, a hybrid apolipoprotein comprised of sequence elements derived from apoE and apoLp-III has been generated [30
]. Studies revealed that this engineered hybrid apolipoprotein adopts a folded protein structure that manifests biological activity of the parent proteins. To further pursue hybrid apolipoprotein research, EPL has been employed to generate a protein hybrid comprised of apoE residues 1-111 and Asp1Cys-substituted apoLp-III residues 1-91. To achieve this, the pelB bacterial expression system was employed to generate Asp1Cys apoLp-III(1-91) for use in ligation studies with C-terminal thiol-adducted human apoE(1-111) derived from an intein fusion protein. Optimization studies to determine conditions that promote protein ligation revealed effects of temperature, pH and thiol agent. The approach described expands the strategies available for EPL and provides a means to specifically modify sequence elements within a novel hybrid apolipoprotein.