title: Secreted production of collagen-inspired gel-forming polymers with high thermal stability in Pichia pastoris.
abstract: Previously, we have shown that gel-forming triblock proteins, consisting of random coil middle blocks and trimer-forming (Pro-Gly-Pro)(9) end blocks, are efficiently produced and secreted by the yeast Pichia pastoris. These end blocks had a melting temperature (T(m)) of ∼41°C (at 1.1 mM of protein). The present work reveals that an increase of T(m) to ∼74°C, obtained by extension of the end blocks to (Pro-Gly-Pro)(16), resulted in a five times lower yield and partial endoproteolytic degradation of the protein. A possible cause could be that the higher thermostability of the longer (Pro-Gly-Pro)(16) trimers leads to a higher incidence of trimers in the cell, and that this disturbs secretion of the protein. Alternatively, the increased length of the proline-rich (Pro-Gly-Pro)(n) domain may negatively influence ribosomal translation, or may result in, for example, hydrophobic aggregation or membrane-active behavior owing to the greater number of closely placed proline residues. To discriminate between these possibilities, we studied the production of molecules with randomized end blocks that are unable to form triple helices. The codon- and amino acid composition of the genes and proteins, respectively, remained unchanged. As these nontrimerizing molecules were secreted intact and at high yield, we conclude that the impaired secretion and partial degradation of the triblock with (Pro-Gly-Pro)(16) end blocks was triggered by the occurrence of intracellular triple helices. This degradation was overcome by using a yapsin 1 protease disruptant, and the intact secreted polymer was capable of forming self-supporting gels of high thermal stability.
authors.affiliation: Wageningen UR Food & Biobased Research, Bornse Weilanden 9, NL-6708 WG Wageningen, The Netherlands. catarinaifsilva@gmail.com
authors.forename: Catarina I F
authors.initials: CI
authors.lastname: Silva
authors.forename: Helena
authors.initials: H
authors.lastname: Teles
authors.forename: Antoine P H A
authors.initials: AP
authors.lastname: Moers
authors.forename: Gerrit
authors.initials: G
authors.lastname: Eggink
authors.forename: Frits A
authors.initials: FA
authors.lastname: de Wolf
authors.forename: Marc W T
authors.initials: MW
authors.lastname: Werten
mesh_terms: D001704:Biopolymers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003094:Collagen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005782:Gels / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability; D044366:Transition Temperature
chemical_list: D001704:Biopolymers; D005782:Gels; D003094:Collagen