Cloning of a novel ovalbumin gene from quail oviduct and its heterologous expression in Pichia pastoris

Abstract

An ovalbumin gene was cloned from Chinese quail (Coturnix coturnix) oviduct by RT-PCR and then inserted into the P. pastoris genome under the control of the methanol inducible 5' alcohol oxidase (AOX) promoter. The recombinant P. pastoris strain was demonstrated to be able to efficiently secrete quail ovalbumin by ELISA analysis using a polyclonal antibody raised against quail ovalbumin. The results showed that induction by 0.75% methanol for 48 h led to the synthesis of secreted quail ovalbumin up to a yield of 5.45 g l(-1). The recombinant ovalbumin was further purified into homogeneity through ion exchange and gel filtration chromatography and SDS-PAGE analysis revealed that, compared to natural ovalbumin, the recombinant ovalbumin could have been glycosylated to the similar extent by P. pastoris.

Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris

“…P. pastoris emerged as successful host for the expression of various heterologous proteins (enzymes and therapeutic proteins) because of the advantages of ease of genetic manipulation, high yield expression of correctly folded proteins, and tightly regulated AOX1 promoter as compared other fungi and other yeast expression systems . Heterologous expression of both inhibitory and noninhibitory serine protease inhibitor family of proteins in P. pastoris is well reported . However, there is no reported literature for the expression of recombinant oAGT or other AGT in P. pastoris .…”

Molecular cloning, expression, and regulation of the ovalbumin gene in pigeon oviduct epithelial cells

“…The pigeon OVA gene displays homology to those of chicken, quail, and emu (McReynolds et al, 1978;Yang et al, 2009;Maehashi et al, 2010). The ovalbumin was a 38-amino acid peptide.…”

“…According to our computational analysis (ProtComp Version 9.0, Softberry), it is an extracellular secretory protein with 3 transmembrane segment residues: 27-47, 233-252, and 292-309. As in quail, pigeon OVA gene transmembrane segments may be involved in the membrane translocation machinery, which facilitates transport across the membrane (Yang et al, 2009). As a SERPIN, pigeon OVA also contains an RCL, which is required for its inhibitory function.…”

“…However, OVA does not inhibit protease activity because it fails to undergo the conformational change known as the "stressed to relaxed transition" (Gettins, 1989(Gettins, , 2002. OVA has been identified in a number of avian species, including chicken, emu, and quail (McReynolds et al, 1978;Yang et al, 2009;Maehashi et al, 2010). Chicken OVA, which is secreted from oviduct tubular gland cells, does not contain an N-terminal leader sequence (McReynolds et al, 1978;Robinson et al, 1986).…”

Precision Fermentation as an Alternative to Animal Protein, a Review

“…Efforts to recombinantly express the proteins present in egg white, in particular chicken ovolabumin (in E. coli, S. cerevisiae or K. phaffii) have been carried out since the 70s and 80s, but without concrete data on productivity, or with extremely low production levels (few mg/L). However, Yang and colleagues achieved an impressive production of 5.45 g/L of quail ovalbumin using K. phaffii [107] (Table 2). Chicken ovotransferrin, the second most abundant protein in egg white, was also expressed using this yeast, with yields of approximately 0.1 g/L (Table 2).…”