ABSTRACT
A simple and efficient way to covalently couple proteins makes use of the natural reactivity of the amino acid side chains exposed at their surface. Methods have been successfully developed for bioconjugation of VNPs with many compounds. The reader might find it useful to consult Refs. [11, 12] for recent overviews. In attempts to obtain a positional control of enzymes on VNPs, the amino acid reactivity needs to be strictly controlled. Many examples show that a direct-ed bioconjugation through amino acids pre-existing on the protein partners is difficult. Although primary amines display high reactiv-ity toward N-hydroxysuccinimide (NHS) esters or isothiocyanates, the large number of lysine residues usually present in the protein prevents a positional control of the modification. Similarly, the use of carbodiimides and amines to capture carboxylates is precluded. More chemoselective are thiol functions. Cysteine residues can be introduced at specific positions within the protein and subsequently alkylated with various maleimides or bromo/iodo acetamides derivatives. The peculiar reactivity of tyrosine can lead to useful alterna-tives and deserves to be discussed. 2.1.1.1.1 CysteineIn the ideal situation, selective coupling may be induced by introducing a specific functional group at a chosen position on each of the two partners (capsid protein [CP] and enzyme). If, for instance, each of the two proteins can be efficiently expressed in Escherichia coli, site-directed mutagenesis can be applied to introduce infrequent amino acids at suitable sites. Owing to the low abundance of cysteine in most protein sequences, disulfide bridges provide the most common way to achieve site-selective protein coupling. However, assigning a cysteine carrying a thiol function on each side provides a homobifonctional system, which suffers from several drawbacks. First, disulfide bridge formation is governed by oxidative conditions often difficult to control, especially if the protein system of interest is only stable in an alkaline range of pH 8-9, where disulfide formation is favored. Second, coupling will give rise to both heterodimerization
(VNP-enzyme) and homodimerization. A strategy to avoid this later requires activation of the thiol, preventing disulfide bridge formation. o-Mesitylenesulfonylhydroxylamine (MSH) is a potential reagent in the oxidative elimination of Cys to dehydroalanine (Dha). To achieve bioconjugation, a disulfide bridge between the Dha moiety on the enzyme and a free cysteine on the VP surface can be subsequently formed (Fig. 2.3). Although oxidative elimination of cysteine is expected to be faster than methionine since the former
proceeds through the removal of an acidic a proton, while the latter through the less labile b proton, methionine reacts with MSH also. When the Dha derivative and the partner bringing the free thiol are mixed, a disulfide crosslinking is favored and methionine residues are regenerated. The end product analysis shows that the method does not induce significant modifications of methionine residues
[13]. In case there is no available cysteine accessible on the VP surface, N-succinimidyl S-acetylthioacetate (SATA), a thiolation reagent, can be used to introduce a protected thiol group on primary amino groups (lysine side chains). Just before addition, a hydroxylamine hydrochloride treatment allows to deprotect the introduced thiol, which is subsequently used for conjugation [14] (Fig. 2.4).
