Secreted production of an elastin-like polypeptide by Pichia pastoris

Abstract

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fedbatch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium.

Expression of synthetic human tropoelastin (hTE) protein inNicotiana tabacum

“…This problem was overcame by using recombinant DNA technologies (Schipperus et al, 2009). However many attempts have been carried out to produce elastin sequence-containing proteins (Floss et al, 2010), our research is the first time to express and produce full human tropoelastin protein (hTE) from plant cells.…”

“…These technologies allowed us to produce high amounts of recombinant proteins within few weeks (Schipperus et al, 2009). In case of tropoelastin protein, by the use of transient expression system based on syringe agroinfiltration through pCambia1390-35S binary vector, we was able to adapt the system to produce human tropoelastin (hTE) protein in Nicotiana tabacum plant in a reduced period of time.…”

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

“…The presence of alternating hydrophobic (Phe, Trp) and hydrophilic (Gln, Arg and Glu) side-chains renders one surface of the ␤-sheet more hydrophilic than the other (Aggeli et al, 1997b). At a peptide concentration of about 0.1 mM, in water, P 11 -2 associates into long, stable semiflexible ␤-sheet ribbons 2005), silk-like proteins (Werten et al, 2008), elastin-like proteins (Schipperus et al, 2009), and self-assembling block copolymers (Martens et al, 2009;Werten et al, 2009), mostly at g l −1 levels. P. pastoris is an attractive host for large-scale production, as it grows in low-cost media, offers good genetic stability, and permits scale-up of the production process without loss of yield (Cregg et al, 1993;Romanos, 1995).…”

Elastomeric Polypeptides

“…An advantage of chemical synthesis is, however, that it enables the facile introduction of functional residues in the polypeptide [27]. In the biosynthetic approach, protein expression in E. coli [27], yeast [28, 29], and plants [30, 31] have been employed. This approach requires the construction of genes encoding for these repetitive polypeptides.…”