Seeding Protein Crystallization with Cross-Linked Protein Crystals

Abstract

Protein crystallization is of great importance because protein crystals have a number of different important applications, including large-scale purification of proteins, determination of protein structure, nanoparticle preparation, and theoretical studies of crystallization. An approach often used to efficiently crystallize proteins is the use of nucleants or seeds (small fragments of protein crystals) that can help increase the probability of protein crystallization. Due to the very positive effect that seeding has on protein crystallization, seeds are now widely accepted and utilized in practical protein crystallization. Here, we show that cross-linked protein crystals (CLPCs), which retain the crystal structure but are much more stable than noncross-linked crystals, can also be used as a new type of seed for promoting protein crystallization. Seeding with CLPCs has effects on both the reproducibility and screening of protein crystals and could improve the optical perfection (well-defined facets) of protein crystals and the probability of obtaining protein crystals. In addition, the cross-linked protein crystals may reduce the concentration of protein molecules needed to obtain protein crystals. Furthermore, CLPCs are very stable in air, and no protective medium is necessary for the long-term storage of CLPCs. This feature makes the CLPC seeding method a potentially powerful technique in practical protein crystallization on either a laboratorial or an industrial scale.

Comparison of the Quality of Protein Crystals Grown by CLPC Seeds Method

“…HR3-267), and the volume of the reservoir was 60 µL. The crystallization temperature was maintained at 293 K. The preparation method of CLPC seeds (seeds 1, seeds 2, and seeds 3) was described in our previous report [29]. Three different CLPC seeds were used in this work.…”

Can (We Make) Bacillus thuringiensis Crystallize More Than Its Toxins?

“…The possibility of using protein crystals as a polyvalent nanomaterial able to concentrate, stabilize, and protect functional proteins has been envisaged for a large range of applications, notably including biosensing (i.e., detection of relevant molecules by the use of biological macromolecules), biotemplating (i.e., assembly of inorganic nanostructures guided by the protein crystal scaffold), catalysis, and vaccine/drug delivery [ 3 , 4 , 5 , 6 ]. Extensive engineering efforts have been made by acting on the crystal packing interfaces, notably through cross-linking [ 89 , 90 ], to tailor the crystal properties and create new scaffolds leading to potentially new crystal functionalities but also by finely tuning the pore structure and associated physicochemical characteristics to adjust the properties of the cargo protein [ 6 ].…”

Production of Cross-Linked Lipase Crystals at a Preparative Scale

“… 23 , 24 Still, at the academic level, there is a renaissance in the interest in the CLEC technology, probably boosted by the extensive knowledge gained during the last two decades in protein purification and crystallization, highly decreasing its cost and laboriousness. 20 , 25 − 28 From the pharmaceutical point of view, the most relevant product, already approved by the US FDA, is the TheraCLEC-Total (Anthera Pharmaceuticals), a mixture of CLECs of lipase and protease and CLEAs of amylase, which is administered orally for the treatment of patients with digestive disorders (celiac disease, cystic fibrosis, etc.). 29 …”

“…Despite the outstanding characteristics of CLECs, such as high operational stability, ease of recycling, or high catalyst and volumetric productivities, the need to crystallize the enzyme has moved the focus to a much easier preparation product known as cross-linked enzyme aggregates (CLEACs), most probably due to the cost and the inherent difficulty of protein crystallization. , Still, at the academic level, there is a renaissance in the interest in the CLEC technology, probably boosted by the extensive knowledge gained during the last two decades in protein purification and crystallization, highly decreasing its cost and laboriousness. ,− From the pharmaceutical point of view, the most relevant product, already approved by the US FDA, is the TheraCLEC-Total (Anthera Pharmaceuticals), a mixture of CLECs of lipase and protease and CLEAs of amylase, which is administered orally for the treatment of patients with digestive disorders (celiac disease, cystic fibrosis, etc.) …”