Use of Carboxypeptidase Y Propeptide as a Fusion Partner for Expression of Small Polypeptides in Escherichia coli

Refolding and Purification of Yeast Carboxypeptidase Y Expressed as Inclusion Bodies in Escherichia coli

“…2A). Utilizing this property, we have developed CPYPR as a fusion partner for the efficient expression of small polypeptides in E. coli (13). Densitometry analysis shows that the CPYPR-His 6 expressed accounts for about 30% of the total cellular protein (Fig.…”

Production and Purification of Recombinant Human Glucagon Overexpressed as Intein Fusion Protein in Escherichia coli

“…Usually glucagon is prepared by microbiological method in composition of hybrid protein, leader part of which provides the transport of polypeptide to Escherichia coli cell periplasm [2] or with the use of yeast Saccharomices cerevisiae [3] followed by hormone isolation from the culture medium. Glucagon biosynthesis to cytoplasm is not used because glucagon can be readily cleaved by proteolytic enzymes this resulting in decrease of the target polypeptide yield, whereas the use of expensive site specific proteases is necessary for its biosynthesis in composition of hybrid protein [4][5][6]. To avoid the difficulties related to cleavage of recombinant protein by various proteases, e.g.…”

“…In this case it is hardly separable from admixtures during further purification. Due to this the methods of both chemical and microbiological synthesis of the polypeptide have been proposed [1][2][3][4][5][6]. Usually glucagon is prepared by microbiological method in composition of hybrid protein, leader part of which provides the transport of polypeptide to Escherichia coli cell periplasm [2] or with the use of yeast Saccharomices cerevisiae [3] followed by hormone isolation from the culture medium.…”

The chromatography‐free release, isolation and purification of recombinant peptide for fibril self‐assembly

“…To optimize carbon utilization during expression, a number of small fusion tags below 20 kDa have been employed for recombinant peptide production. These include ketosteroid isomerase (KSI) (14.4 kDa) (Cipakova et al, 2006), PaP3.30 (17.5 kDa) (Rao et al, 2004), carboxypeptidase Y propeptide (CPY) (10.1 kDa) (Oh et al, 1999), PurF (14 kDa) , truncated GST (14 kDa) (Hu et al, 2008), thioredoxin (Trx) (12.2 kDa) (Huang et al, 2008), and small ubiquitin-like modifier (SUMO) (11.5 kDa) (Bosse-Doenecke et al, 2008). Recent work has demonstrated acceptable expression of peptides thought to be toxic to host bacteria, including human parathyroid hormone (Oh et al, 1999), cecropin (Rao et al, 2004) and the amyloidogenic peptides (Sharpe et al, 2005), as soluble fusions without any apparent adverse effect on the cell (Fu et al, 2005;Lopes et al, 2004;Xu et al, 2007).…”

“…These include ketosteroid isomerase (KSI) (14.4 kDa) (Cipakova et al, 2006), PaP3.30 (17.5 kDa) (Rao et al, 2004), carboxypeptidase Y propeptide (CPY) (10.1 kDa) (Oh et al, 1999), PurF (14 kDa) , truncated GST (14 kDa) (Hu et al, 2008), thioredoxin (Trx) (12.2 kDa) (Huang et al, 2008), and small ubiquitin-like modifier (SUMO) (11.5 kDa) (Bosse-Doenecke et al, 2008). Recent work has demonstrated acceptable expression of peptides thought to be toxic to host bacteria, including human parathyroid hormone (Oh et al, 1999), cecropin (Rao et al, 2004) and the amyloidogenic peptides (Sharpe et al, 2005), as soluble fusions without any apparent adverse effect on the cell (Fu et al, 2005;Lopes et al, 2004;Xu et al, 2007). A commonly used fusion tag is Trx, which has been routinely expressed at levels in excess of 30% of total cell protein (TCP) and has proved to be an excellent and stable solubility enhancer (Huang et al, 2007;LaVallie et al, 1993;Shlyapnikov et al, 2008;Tenno et al, 2004;Zhou et al, 2005).…”