Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris

Abstract

A custom-designed, highly hydrophilic gelatin was produced in Pichia pastoris. Secreted production levels in single-copy transformants were in the range 3-6 g/l of clarified broth and purification to near homogeneity could be accomplished by differential ammonium sulfate precipitation. Despite the fact that gelatins are highly susceptible to proteolysis because of their unfolded structure, the recombinant protein was shown to be fully intact by SDS-PAGE, N-terminal sequencing, gel filtration chromatography and mass spectrometry. Owing to its highly hydrophilic nature, the migration of the synthetic gelatin in SDS-PAGE was severely delayed. Esterification of the carboxylic amino acid side chains resulted in normal migration. The high polarity of the synthetic gelatin also accounts for its negligible surface activity in water at concentrations up to 5% (w/v), as determined by tensiometry. Circular dichroism spectrometry showed that the non-hydroxylated gelatin did not form triple helices at 4 degrees C. The spectrum was even more representative of the random coil conformation than the spectrum of natural non-hydroxylated gelatins.

Close Packing of Listeria monocytogenes ActA, a Natively Unfolded Protein, Enhances F-actin Assembly without Dimerization

“…Although PII helices will not melt out at elevated temperatures, the lack of overall shape change upon heating is probably due to the fact that ActA is mostly random coil, and therefore, there is very little ordered structure to melt out. In addition, the overall shape of the CD spectrum is virtually identical to the CD spectra previously reported for two other natively unfolded proteins (55,56). Analysis of the amino acid sequence of ActA using PONDR (Predictor of Natural Disordered Regions; access provided by Molecular Kinetics (copyright 2004)) predicts ActA to be 74, 100, 89, and 100% disordered using the VLXT (copyright 1999 by the Washington State University Research Foundation (61)), VSL1 (62), XL1_XT (63), and VL3 (64) algorithms, respectively.…”

“…However, ActA continues to migrate with an abnormally high relative mobility even when this polyproline rich domain is deleted (14). There are numerous reports of natively unfolded proteins traveling with an abnormally low relative mobility on SDS-PAGE (55,56). The mechanisms by which proteins bind to SDS are only partially understood.…”

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

“…The banding pattern of low-level degradation products in the kex2 strain was not significantly different from that in the wild type. We previously suggested that these minor fragments might have originated from limited cleavage by Kex2 at Arg residues within a suboptimal sequence context because a tailored gelatin with no Arg residues at all was secreted completely intact (47) and because even the relatively discriminative S 2 and S 4 subsites (Schechter and Berger [42] nomenclature) of S. cerevisiae Kex2 have some leeway in the substrate residues that they can accommodate (8,43). Apparently, however, few sites in Col1a1-2 other than Met-Gly-Pro-Arg meet the Kex2 protease's overall substrate requirements, as most of the minor bands were also present in the kex2 disruptant.…”

“…Traditional animal gelatin is a versatile biomaterial with both medical and technical applications. Gelatins produced in microbial systems provide the added benefit of being well defined, free of animal-derived contagious agents, and suitable for modification for specific needs (35,46,47).…”

“…Removal of the Met-Gly-Pro-Arg motifs from the gene construct by site-directed mutagenesis resulted in the production of a predominantly full-length product and a number of shorter fragments present in lower quantities (46). A customdesigned gelatin with no Arg residues was produced completely intact, so we hypothesized that the minor fragments may have originated from limited Kex2-like proteolysis at suboptimal monoarginylic motifs (47). In addition to a Kex2 homolog, the aspartic yapsin proteases (18,30,36) could also be involved, since their substrate specificities overlap those of Kex2 in S. cerevisiae (10).…”