Gelatins from yeast provide novel possibilities

Recombinant gelatinen (natuurlijk of tailor made) zijn eeen goed alternatief ter voorkoming van prion besmetting die bse veroorzaak

Method for the production of hydroxylated collagen-like compounds using prolyl hydroxylase from Hansenula polymorpha

The present invention relates to a method for the production of collagen-like compounds containing hydroxylated proline residues. Of specific interest is the production of recombinant collagen-like compounds in which hydroxylation of proline residues is achieved by a prolyl hydroxylase from a fungus, preferably a yeast, in particular Hansenula polymorpha. Also the invention concerns a method for controlling the hydroxylation of proline residues by such a prolyl hydroxylase characterised by the addition of collagen-like oligopeptides, such as gelatine hydrolysate, in particular gelatone or peptone

Eiwitpolymeren : monomeervolgorde op bestelling, assemblage op elke schaal

De Biobased-Productsdivisie van AFSG-Wageningen UR gebruikt micro-organismen om op commando polymeren te maken met een van a tot z ontworpen monomeervolgorde en blokstructuu

Production in Pichia pastoris of protein-based polymers with small heterodimer-forming blocks

Some combinations of leucine zipper peptides are capable of forming a-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications

Endogenous prolyl 4-hydroxylation in Hansenula polymorpha and its use for the production of hydroxylated recombinant gelatin

Several yeast systems have recently been developed for the recombinant production of gelatin and collagen. Amino acid sequence-specific prolyl 4-hydroxylation is essential for the gel-forming capacity of gelatin and for the proper folding of (pro)collagen. This post-translational modification is generally considered to be absent in microbial eukaryotic systems and therefore co-expression of heterologous (human or animal) prolyl 4-hydroxylase would be required. However, we found that the well-known protein expression host Hansenula polymorpha unexpectedly does have the endogenous capacity for prolyl 4-hydroxylation. Without co-expression of a heterologous prolyl 4-hydroxylase, both an endogenous collagen-like protein and a heterologously expressed collagen fragment were found to be sequence-specifically hydroxylated

Protein cross-linking tools for the construction of nanomaterials

Across bioengineering there is a need to couple proteins to other proteins, or to peptides. Although traditional chemical conjugations have dominated in the past, more and more highly specific coupling strategies are becoming available that are based on protein engineering. Here we review the use of protein modification approaches such as enzymatic and autocatalytic protein-protein coupling, as well as the use of hetero-dimerizing (or hetero-oligomerizing) modules, applied to the specific case of linking together de novo designed recombinant polypeptides into precisely structured nanomaterials. Such polypeptides are increasingly being investigated for biomedical and other applications. In this review, we describe the protein-engineering based cross-linking strategies that dramatically expand the repertoire of possible molecular structures and, hence, the range of materials that can be produced from them

Secreted production of self-assembling peptides in Pichia pastoris by fusion to an artificial highly hydrophilic protein

The undecapeptides CH3CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH2 (P11-2) and CH3CO-Gln-Gln-Orn-Phe-Orn-Trp-Orn-Phe-Orn-Gln-Gln-NH2 (P11-14) have unique self-assembly characteristics and broad application potential. Originally, these peptides were produced by chemical synthesis, which is costly and difficult to scale up to industrial levels in an economically feasible way. This article describes the efficient secreted production of these peptides (with free termini and ornithines replaced with lysines) in the methylotrophic yeast Pichia pastoris. The peptides were produced as enterokinase-cleavable fusions to the C-terminus of an artificial Solubility-Enhancing Protein (SEP). In vitro, the fused highly hydrophilic SEP proved to prevent self-assembly of the peptides. The SEP domain also facilitates product detection and allows convenient separation of the fusion protein from the broth by simple salt precipitation. After cleavage of the purified fusion protein with enterokinase, the free undecapeptides were obtained and P11-2 spontaneously assembled into a self-supporting gel, as intended. The properties of the SEP carrier could be advantageous for the production of other peptides

Block co-polypeptide and hydrogels made thereof

Disclosed is a block co-polypeptide comprising at least two blocks T and at least one block S, with blocks T and S alternating, wherein each T independently denotes a Trimerizing Block being a polypeptide block capable of forming a thermoreversible collagen-like triple helix structure, and whereineach S independently denotes a Spacer Block S being a polypeptide neither forming a collagen-like triple helix. The block co-polypeptide is capable of forming a gel, and is useful as an animal-free, biocompatible substitute for gelatin. Uniquely, the polypeptide allows the independent controlling of the melting temperature of the gel, the stiffness and crosslink density of the gel, the lower critical gel-forming concentration related to the length of the molecules, and unrelated (e.g. biological, physiological, pharmaceutical or signalling) functions of the molecule