VLAG
Dresselhuis, De Hoog, Van Aken, Cohen Stuart

VLAG
Silletti, Vingerhoeds, Van Aken, Norde

Biosynthesis and properties of new ionic block polypeptides

VLAG
Martens, De Wolf (A&F), de Vries, Cohen Stuart

Block-copolymers have for the past 50 years continued to surprise us with their self assembled and ordered nanostructures which have found many technical and industrial applications. Using the natural protein production machinery of yeast and DNA as a template, we produced pH responsive protein  block-copolymers in which we have control over the length and monomer (amino acid) sequence. Nano-structures of classical block-copolymers result from micro phase separation and depend on the ratio of monomers with different physical properties and their distribution along the polymer chain. In proteins, 21 different amino acids with different physiochemical properties and conformational preferences can be distributed along the chain according to a design, leading to molecules of equal length with defined blocks with defined sequences  of chiral monomers. Within a design, the molecules have the same size and shape, which additional to phase separation, promotes self assembly.

Bio-Block-copolymers
The molecules that we fabricated are 802 amino acids long and can be denoted as ABBA and BAAB. They were produced by fermentation with an exceptionally high production yield of 5 to 8 g/l of cell free fermentation broth. The first four amino acids are no part of a block. The A block is a 207 amino acid long hydrophilic random coil that remains highly water soluble at various pH and salt concentrations. The 192 amino acids long B block is a silk-like glycine (G) alanine (A) repeat which also contains the negatively charged glutamic acid (E): (GAGAGAGE)₂₄. It is soluble at neutral pH but changes into an insoluble β-sheet when charge is neutralized at low pH.
Aggregation of the B blocks in one direction while the A block prevents aggregation in lateral directions leads to self assembly of precisely defined protein fibrils which are 2-5 nm wide and several microns long. Having such high aspect ratio, the fibrils form dilute gels already at 0.3 g/l.  The gels dissolve again when the pH is raised. Gels and materials from these proteins can contribute to developments in material science and in scaffolds for tissue culturing.

VLAG
Lindhoud, Norde, De Vries, Cohen Stuart

Encapsulation of enzymes is very interesting for pharmaceutical, food and industrial applications.  Since proteins are rather fragile structures encapsulation can be a way to protect them, not only from physical (e.g. temperature differences) or chemical influences (e.g. reactive compounds), but also from biological influences (e.g. proteolytic attacks).  We use polyelectrolyte complex micelles for the encapsulation. So far we have been able to prepare stable micelles in which we can control the number of encapsulated proteins.  Currently we are investigating whether we can influence the enzymatic activity of the encapsulated proteins and whether we can release the proteins in a controlled manner.

Collagen inspired self aggregating materials

VLAG
Szezewska, de Wolf, vd Gucht, Cohen Stuart

Collagen is the main component of connective tissues in animals. It takes up to 25% of total protein content in mammalians. The most characteristic feature of collagen is the triple-helical structure. Such kind of arrangement is possible due to a characteristic amino acid sequence. The motif, continuously repeated is [Gly,X1,X2]. Glycine is present every third position and always goes to the interior (axis) of the triple helix. That is because there is not enough space for any larger side groups than hydrogen. Positions X1 and X2 mostly are occupied by proline and hydroxyproline. These two aminoacids thermally stabilize the structure of collagen. Gelatin is a product of partial denaturation (hydrolization) of collagen.

Gelatin and collagen due to their properties have a lot of medical, pharmaceutical and cosmetic applications such as intravenous infusions, matrix implants, drug delivery systems, wound treatment materials etc. In addition gelatin is widely used as a gelling agent in food industry. Being animal derived, traditional gelatin and collagen are subjected to contaminations with prions, animal viruses etc. Beside there is a relatively high rate of allergic reactions.

To overcome problems mentioned above and produce “healthy” and biocompatible gelatin the genetic engineering has been employed. Novel (self–designed) protein can be produced by modified yeasts (Pichia Pastoris).

The single chain of investigated protein consists of 3 blocs. End blocs are identical and their primary sequence is inspired by collagen [Gly,X1,X2]. Hence under proper conditions they can assemble together and form triple helices. As a consequence, the physical gel is obtained.

From application point of view, physico-chemical properties of the gel are crucial. In order to be able to control them, the gelation (melting) mechanism and internal structure of the system have to be deepened and understood.
This project is focused on the physico-chemical characterization of gels obtained from recombinant proteins. The techniques used are rheology, light scattering, circular dichroism  and calorimetry.