Large-scale protein preparation

Protocol 1 Large-Scale Protein Preparation from Yeast.163... 

RPLC is the most common technique for small organic molecules. It is also a powerful tool for preparative peptide separations, but is less common for protein purification, because it is often associated with protein denatura-tion. Nevertheless, there are successful examples of large-scale protein purification by RPLC [99], In this thesis the substances used and produced in the biotechnological synthesis, in papers I and II, were small molecules (Mw < 1000) and therefore belong to a sample class for which RPLC is the dominant separation technique. 

Endo and co-workers at Ehime University, Matsuyama, Japan, have led the development of the most promising eukaryotic cell-free system to date, based on wheat embryos. A significant advance made by this group was the development of pEU expression vectors that have overcome many of the difficulties associated with mRNA synthesis for translation in a eukaryotic system [8]. In addition to extensive optimization of reaction conditions that have seen improvements in protein synthesis rates, Endo and colleagues have improved wheat extract embryo preparation protocols to enhance the stability of these systems to a remarkable extent [9]. When coupled with the dialysis mode of reaction, Endo et al. were able to maintain translational activity in a coupled transcription/ translation wheat embryo reaction for 150 hours, producing 5 mg of enzymatically active protein per mb reaction mixture [10]. This again represents a serious alternative to in vivo methods of large-scale protein production. 

Several peptides with specific properties may be prepared from milk proteins, either in vivo or in vitro some may have commercial potential and can be produced on a relatively large scale by preparative ion-exchange chromatography. 

For every protein purification problem there is always an affinity solution, but cost and safety considerations may render these solutions impractical. As an example, antibodies are widely used for analysis, where only relatively small amounts are usually required, but their production and purification on a large scale for preparative-scale chromatography may be difficult to justify economically. In some cases, Hhybridoma technology may be able to address this problem. Even if production costs are acceptable, the immobilized antibodies may be unstable over the sequence of sample application, elution, and sanitation required for multiple use of the affinity adsorbent. For these reasons, while biological ligands (antibodies, enzymes, receptors, lectin. 

The introduction of delayed ion extraction in MALDI-TOF instruments in 1995 resulted in a tremendous improvement in analytical performance, mass resolution and accuracy. Using this technique, peptide mass mapping gained high specificity for protein identification and an ability to identify individual components among simple protein mixtures [60, 61]. Improved sample preparation methods also provided an enhanced sensitivity to enable analysis of femtomolar levels of protein [5, 19], Today, the automation of MALDI-MS data acquisition enables the analysis of hundreds of samples each day in large-scale protein identification experiments [62]. 

History. Methods for the fractionation of plasma were developed as a contribution to the U.S. war effort in the 1940s (2). Following pubHcation of a seminal treatise on the physical chemistry of proteins (3), a research group was estabUshed which was subsequendy commissioned to develop a blood volume expander for the treatment of military casualties. Process methods were developed for the preparation of a stable, physiologically acceptable solution of alburnin [103218-45-7] the principal osmotic protein in blood. Eady preparations, derived from equine and bovine plasma, caused allergic reactions when tested in humans and were replaced by products obtained from human plasma (4). Process studies were stiU being carried out in the pilot-plant laboratory at Harvard in December 1941 when the small supply of experimental product was mshed to Hawaii to treat casualties at the U.S. naval base at Pead Harbor. On January 5, 1942 the decision was made to embark on large-scale manufacture at a number of U.S. pharmaceutical plants (4,5). 

Mammalian cells are commonly employed for the production of therapeutic and diagnostic proteins, since they are able to correctly synthetize the large and complex structures that the human body requires as medicine [1]. Nowadays, they are employed for the large-scale production of recombinant therapeutic proteins, monoclonal antibodies (MAbs) and viruses used in the preparation of vaccines (e.g. against rabies, hepathytis B, polio, etc) [2]. An overview of some licensed/approved products derived from mammalian cell culture is given in Table 1. 

Lokra, S., Helland, M. H., Claussen, I. C., Straetkvern, K. O., Egelandsdal, B.10.1016/j.lwt.2007.07.006 (2007). Chemical characterization and functional properties of a potato protein concentrate prepared by large-scale expanded bed adsorption chromatography. LWT - Food Sci. Technol. 

Today, it is well-known that peptides or proteins exhibit various kinds of taste. Our group has been researching on the relationship between taste and structure of peptides, BPIa (Bitter peptide la, Arg-Gly-Pro-Pro-Phe-Ile-Val) (7 as a bitter peptide, Om-p-Ala-HCl (OBA), Om-Tau-HCl as salty peptides(2j, and "Inverted-Aspartame-Type Sweetener" (Ac-Phe-Lys-OH) as a sweet peptide(5). The relationship between taste and chemical structure was partly made clear. Since commercial demand for these flavor peptides is increasing, we need to develop new synthetic methods which can prepare these peptides in large scale. We developed the following two methods (1) protein recombination method as a chemical method, (2) enzymatic synthesis using chemically modified enzyme as a biochemical method. 

Janson JC, Peterson T (1993) Large scale chromatography of proteins. In Ganetsos G, Barker PE (ed) Preparative and production scale chromatography. Marcel Dekker, New York, p 559... 

---