Construction of a synthetic protein using PCR with a high essential amino acid content for nutritional purposes.

Ovalbumin is considered a protein of high nutritional value because it contains essential amino acids and is highly digestible. Therefore, it has a high biological value. Currently, the high food demand requires worldwide attention because food production is insufficient. Therefore, other alternatives are necessary to satisfy food demands, such as protein engineering. In this work, a protein with a high essential amino acid content similar to ovalbumin was synthesized by protein engineering, expressed, and digested in vitro. The assembly and sequential overlap extension PCR strategy was used to synthesize a 345-bp gene that encodes a high essential amino acid content protein (HEAAP). The 345-bp product was cloned into the vector pBAD TOPO®, and expressed in Escherichia coli BL21. PCR reactions and sequencing demonstrated the presence, orientation, and correct sequence of the insert. HEAAP expression was induced by L-arabinose and then purified using Ni-NTA affinity chromatography. The expression in E. coli was low and barely detected by Western blot assay. The in vitro multienzyme digestibility of HEAAP was around 79%, which suggests that the protein is potentially nutritious. Virtual analysis classifies the protein as unstable and hydrophilic, with a half-life in E. coli of 10 h. The recombinant HEAAP was successfully synthesized, but it is necessary to improve the digestibility and to optimize expression including selecting other expression models.

Chronic improvement of amino acid nutrition stimulates initiation of global messenger ribonucleic acid translation in tissues of sheep without affecting protein elongation.

Initiation of mRNA translation and elongation of the polypeptide chain are 2 regulated processes responsible for the short-term postprandial acceleration of protein synthesis in animal tissues. It is known that a chronic increase in the absorptive supply of AA stimulates protein synthesis in ruminant animals, but effects on translation initiation and elongation are unknown. To determine whether initiation or elongation phases of global mRNA translation are affected by chronic elevation of AA supply, 24 ewe lambs of 25.9 +/- 2.5 kg of BW were randomly allocated to 4 treatment groups of 6 lambs each. All lambs received a basal diet of barley and hay at 1.2 times maintenance ME intake. Treatments were an intravenous (i.v.) saline infusion as a control, i.v. infusion of 6 essential AA (EAA; Arg, Lys, His, Thr, Met, Cys) for 10 d, i.v. infusion of the same EAA excluding Met and Cys (EAA-SAA) for 10 d, and an oral drench of fishmeal twice daily for 17 d. Fishmeal supplementation supplied an extra 719 mg of N x kg(-0.75) x d(-1) and N retention was increased 519 mg x kg(-0.75) x d(-1) over the control. The EAA treatment supplied an extra 343 mg of N x kg(-0.75) x d(-1) directly into the blood, and N balance was increased by 268 mg x kg(-0.75) x d(-1). Deletion of Met plus Cys from EAA had no effect on N balance. The results indicate that Met plus Cys did not limit body protein gain on the basal diet alone or the basal diet plus 6 AA. Protein fractional synthesis rates in liver, duodenum, skin, rumen, semimembranosus, and LM were measured by a flooding dose procedure using L-[ring-2,6-(3)H]-Phe. Ribosome transit times were estimated from the ratio of nascent to total protein-bound radioactivities. Fishmeal and EAA treatments had no effect on RNA, DNA, or protein contents of tissues, but fractional synthesis rate, translational efficiency, and concentrations of active ribosomes were consistently elevated. Ribosome transit time was not affected by long-term AA supply. We conclude that the chronic stimulation of protein synthesis by long-term i.v. infusion of EAA or supplementation with an undegradable protein source is brought about by an improvement in the rate of initiation of mRNA translation with no change in the rate of polypeptide chain elongation.

Functional properties of whey, whey components, and essential amino acids: mechanisms underlying health benefits for active people (review).

Whey proteins and amino acid supplements have a strong position in the sports nutrition market based on the purported quality of proteins and amino acids they provide. Recent studies employing stable isotope methodology demonstrate the ability of whey proteins or amino acid mixtures of similar composition to promote whole body and muscle protein synthesis. Other developing avenues of research explore health benefits of whey that extend beyond protein and basic nutrition. Many bioactive components derived from whey are under study for their ability to offer specific health benefits. These functions are being investigated predominantly in tissue culture systems and animal models. The capacity of these compounds to modulate adiposity, and to enhance immune function and anti-oxidant activity presents new applications potentially suited to the needs of those individuals with active lifestyles. This paper will review the recent literature that describes functional properties of essential amino acids, whey proteins, whey-derived minerals and other compounds and the mechanisms by which they may confer benefits to active people in the context that exercise is a form of metabolic stress. The response to this stress can be positive, as with the accretion of more muscle and improved functionality or greater strength. However, overall benefits may be compromised if immune function or general health is challenged in response to the stress. From a mechanistic standpoint, whey proteins, their composite amino acids, and/or associated compounds may be able to provide substrate and bioactive components to extend the overall benefits of physical activity.

Taurine: a conditionally essential amino acid in humans? An overview in health and disease.

Taurine, a sulphur containing amino acid, is the most abundant intracellular amino acid in humans, and is implicated in numerous biological and physiological functions. This comprehensive overview explores areas, from its characterisation to its potential clinical benefit as a conditionally essential amino acid and a pharmaconutrient. In healthy individuals the diet is the usual source of taurine; although in the presence of vitamin B6 it is also synthesised from methionine and cysteine. Taurine has a unique chemical structure that implies important physiological functions: bile acid conjugation and cholestasis prevention, antiarrhythmic/inotropic/chronotropic effects, central nervous system neuromodulation, retinal development and function, endocrine/metabolic effects and antioxidant/antiinflammatory properties. Taurine is an essential amino acid for preterm neonates and is assured by breast milk. Specific groups of individuals are at risk for taurine deficiency and may benefit from supplementation, e.g. patients requiring long-term parenteral nutrition (including premature and newborn infants); those with chronic hepatic, heart or renal failure. Further studies are required to determine the benefits of replenishing taurine pools as well as the need to include taurine routinely in parenteral nutrition regimens.