Cross-linked protein crystals (CLPCs) constitute a novel type of molecular sieves with high porosity. In order to characterize the fully hydrated CLPC, the method of macromolecular porosimetry was applied. This technique allows one to estimate the apparent pore sizes and pore size distribution in solid and soft hydrated porous sorbents directly from size exclusion chromatography. According to this method, CLPCs offer a wide range of pore size (15−100 Å), porosity (0.5−0.8), and pore surface area (800−2000 m2/g). These CLPC materials can be made chemically and mechanically stable, and are capable of separating molecules by size, chemical structure, and chirality.

The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present paper, we propose the use of cross-linked protein crystals (CLPCs) as antigens. We compare the immunogenicity of CLPCs of human serum albumin with that of soluble protein and conclude that there are marked differences in the immune response to the different forms of human serum albumin. Relative to the soluble protein, crystalline forms induce and sustain over almost a 6-month study a 6-to 10-fold increase in antibody titer for highly cross-linked crystals and an approximately 30-fold increase for lightly cross-linked crystals. We hypothesize that the depot effect, the particulate structure of CLPCs, and highly repetitive nature of protein crystals may play roles in the enhanced production of circulating antibodies. Several features of CLPCs, such as their remarkable stability, purity, biodegradability, and ease of manufacturing, make them highly attractive for vaccine formulations. This work paves the way for a systematic study of protein crystallinity and cross-linking on enhancement of humoral and T cell responses.

No abstract

Horse liver alcohol dehydrogenase was crystallized in the presence of its cofactor. The cross-linked enzyme crystals (CLECs) produced showed good catalytic activity without the addition of extra cofactor (see scheme). The enantioselectivity and stereochemical preference of the CLEC were the same as with the soluble enzyme, and both cofactor and enzyme were considerably more stable in CLEC form. Cofactor regeneration studies on the reduction of cinnemaldehyde indicate the potential for a high level of catalyst productivity.

The use of dehydrogenases in organic synthesis is often limited by the intrinsic instability of enzymes and their nicotinamide cofactors. [1] The protein part of the molecule can be efficiently stabilized by several techniques such as directed evolution, [2] immobilization, [3] and protein crystallization and cross-linking. [4] The latter approach has turned out to be especially efficient in producing robust and productive biocatalysts for chemical synthesis. [5] Here, we expand this approach to the stabilization of the cofactor part of the dehydrogenase molecule. Horse liver alcohol dehydrogenase (HLADH) was crystallized in the presence of reduced nicotinamide adenine dinucleotide (NADH), and the resulting crystals were treated with glutaraldehyde to yield the cross-linked enzyme crystals (CLECs). The crystallized and cross-linked HLADH was first introduced by Lee et al., and it demonstrated good activity (26 % of that in solution) and an increased stability of the cross-linked crystals in the presence of zinc salts. [6] In this work, we use this system to address two main questions: 1) Is a cofactor more stable when bound inside the enzyme crystal? 2) Is it possible to regenerate a cofactor using a coupled substrate system, thus making HLADH-NADH-CLEC a useful catalyst for organic synthesis?The activity of soluble enzyme and various HLADH-CLEC preparations was compared in the reduction of 6-methyl-5hepten-2-one (1) in the presence of isopropanol for cofactor regeneration (Scheme 1). The results, presented in Table 1, afford several conclusions. The HLADH-NADH-CLECs exhibit higher activity when HLADH is cocrystallized with a cofactor and an inhibitor, DMSO. In this case, the resulting complex exhibited 64 % of the activity of the soluble enzyme in the absence of an exogenous cofactor. DMSO seems to be excellent agreement to those of a natural sample ( 1 H and 13 C NMR, IR, TLC, [a] 20 D 13.0 (c 1.09, MeOH). In conclusion, this total synthesis of ()-discodermolide proceeds in 27 steps and 7.7 % overall yield for the longest linear sequence starting from commercial methyl (S)-3hydroxy-2-methylpropionate. The three key subunits were synthesized efficiently using boron-mediated anti-selective aldol reactions of chiral ketones (S)-6, (S)-11, and (S)-17. This synthesis has the potential to provide useful quantities of ()discodermolide, which will allow detailed biological evaluation, as well as offering a variety of options for analogue chemistry.