In addition to a role in nutrition, proteins may have further health benefits due to the release by proteolysis of encrypted bioactive peptides (BPs). It has been widely demonstrated that BPs are intervention agents against chronic human diseases such as hypertension and for maintaining general well-being (Hartmann & Meisel, 2007; De Leo et al., 2009; Udenigwe & Aluko, 2012). The use of BPs allows for a number of advantages in comparison with chemical drugs, as they come from natural sources and are therefore safer than synthetic molecules. Although proteins of animal and plant origins, such as milk, fish, cereals, eggs, muscles, and peas, have been exploited as relevant sources for BPs production (Roy et al., 2010; Gomez-Guillen et al., 2011; Holdt & Kraan, 2011; Ryan et al., 2011; Urista et al., 2011; Lico et al., 2012), since extraction of BPs from proteins of plant and animal origin is difficult and costly, companies should be encouraged to invest in new methods that lead to cheaper recovery. BPs are considered to be key ingredients in functional foods, which can be used for struggling against chronic diseases (Korhonen, 2009). These are classified according to their physiological activity as antihypertensive (Jiang et al., 2010), antimicrobial (Singh & Singh, 2011; Espitia et al., 2012), antioxidative (Sarmadi & Ismail, 2010; Coda et al., 2012), antitumor (Rizzello et al., 2012), mineral-binding (Berrocal et al., 1989), and opiate-like compounds (Varamini et al., 2012), among others. The most widely studied peptides are those belonging to the antihypertensive group, which are capable of inhibiting the angiotensin-I converting enzyme (ACE, EC 18.104.22.168), a type-I membrane anchored dipeptidyl carboxypeptidase that is essential for blood pressure regulation and electrolyte homeostasis through the renin-angiotensin system (Hernandez-Ledesma et al., 2011; Phelan & Kerins, 2011). Three ACE-inhibitory BPs arising from bovine β-casein have been characterized (Gobbetti et al., 2000; Minervini et al., 2003). These compounds, named BP1, BP2, and BP3, correspond, to fragments 57–66, 73–82, and 47–52 of bovine β-casein (A2 allelic variant), respectively. The pro-active precursors of these BPs (termed as Pro-BP1, Pro-BP2, and Pro-BP3) were previously expressed as fusion proteins in Escherichia coli cells, using the GST expression system. The pro-active precursors were generated by the addition of two fragments consisting of five amino acids, at N- and C-term of each BP. After expression in E. coli as fusion proteins with the GST protein, BP1, BP2, and BP3 were released from Pro-BPs using partially purified membrane proteinases isolated from Lactobacillus helveticus PR4 (Losacco et al., 2007).
In this work, synthetic genes encoding BPs and related Pro-BPs pro-active precursors were designed, cloned and expressed using pAM1, an E. coli-Bifidobacterium shuttle vector. The recombinant plasmids isolated from E. coli clones were then electrotransferred into the probiotic strain Bifidobacterium pseudocatenulatum M115. Expression of the cloned synthetic sequences was assessed by reverse transcription PCR (RT-PCR) analysis, while correct activation of the BPs released in cell-free cellular lysates of the transformed strains was evaluated by the analysis of their ACE-inhibitory activity.