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.

Routes towards Novel Collagen-Like Biomaterials

“…Synthetic collagen-based block copolymers are artificially designed constructs and combine some of the approaches mentioned so far. When short collagen-like (GPP) n -peptides are positioned around a hydrophilic unstructured core domain and expressed in P. pastoris, a stable gelatin-like hydrogel can be produced with high yields [77,78]. Since these structures, like engineered collagens, are based on synthetic genes, the addition of short peptide sequences such as cell adhesion motifs or calcification sites is trivial.…”

Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115

“…PTH (1-84) was also susceptible to yapsins when expressed in S. cerevisiae and the use of multiple-yapsin-deficient mutant was efficient in preventing the proteolytic degradation [5]. YPS1 gene of P. pastoris was first cloned and characterised by Werten and Wolf [24], and the authors found that the yps1 -disrupted P. pastoris strain was beneficial for secreting production of collagen-inspired gel-forming polymers [20]. Besides, Yao et al [26] found that the significant reduction of HSA-AK15 (R13 K) degradation, which occurred in the sequence of AK15 (R13 K), was achieved by the YPS1 disruption in P. pastoris strain.…”

Production of protein‐based polymers in Pichia pastoris

“…Owing to their high proline content, the end blocks are sufficiently stable to form triple helices in the absence of prolyl-4-hydroxylation (Section 9.1.2). The melting point of (Pro-Gly-Pro)n peptides can be controlled by their length n. Thus, besides T9 end blocks, we also constructed Tn end blocks with n = 6, 12, and 16 [114,115]. The proteins with n = 6 or 9 were produced at several g/L of cell-free broth, and the proteins with n = 12 or 16 at 0.6 and 0.4 g/L, respectively.…”

“…polymers, potentially capable of forming triple helices close to the Glu-Ala repeats, do show low levels of extended species (Chapter 6) [114]. Overexpression of DPAPase A in S. cerevisiae led to increased maturation of α-factor pheromone [163], which suggests this may be a viable strategy for improving the processing of the ppαF leader when used to drive secretion of heterologous proteins.…”

“…Alternatively, it could be argued that the increased length of the proline-rich (Pro-Gly-Pro)n stretches may negatively impact ribosomal translation, or may, through increased hydrophobic interactions, result in protein aggregation. To discriminate between these possibilities, we produced molecules with randomized end blocks and otherwise unchanged amino acid composition [114]. The fact that these, by definition nontrimerizing, molecules were secreted at high yield, shows that indeed trimer-formation disturbs secretion.…”