Yeasts yeast protein extraction

The limited data indicate that yeast proteins extracted by the current, more conventional methods lack the requisite functional properties for many applications. 

In this chapter, we described an easily implemented, effective, and highly reproducible dual-column HPLC prefractionation method that we have developed for improving routine proteomic analyses. The approach results in multifold increases in the numbers of proteins that can be confidently identified by LC-MS of whole cell lysates without fractionation. We outline the key steps in the overall procedure, from sample preparation through to MS/MS and attendant data analysis, using yeast soluble protein extract as a test mixture. 

The use of MS-MS to provide sequence information has been described [13] for the study of proteins extracted from yeast (Saccharomyces cerevisiae). The procedure was somewhat complex and consisted of the following steps ... 

Figure 5.18 Silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae. From Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and KaLkM-nen, N., Rapid Commun. Mass Spectrom., 15, 1685-1692, Copyright 2001. John WUey Sons Limited. Reproduced with permission.

Figure 1.2 Schematic outlining the purification protocol. The principal steps of purifying FLAG-tagged eIF2B proteins from yeast whole cell extracts are outlined in the diagram for details, refer to the main text.

Western-blot analysis of yeast cell extracts shows a 72 kDa protein with rabbit anti-(A saitoi carboxypeptidase) serum, which is consistent with the apparent molecular mass of the native A. saitoi carboxypeptidase. Conversely, the extracts obtained from yeast cells transfected with the vector (pG-3) alone or with cDNA in reverse orientation (pGP31), as negative controls, yielded no stainable protein. 

Specific modifications of proteins result from adding a selected reagent to the pure protein or crude protein-rich material. This may be done in the course of a fundamental study in protein chemistry or as a step in the production of a bulk protein product for practical purposes. The same chemical modification can be useful in both processes. For example, enzyme chemists use charge-changing modifications to dissociate oligomeric proteins to their monomer components, while the same modifications are proposed as a means of solubilizing yeast proteins to permit their extraction for use in foods (11). This chapter is concerned mainly with the many types of intended modifications. 

Isolation of Proteins with a Reduced Nucleic Acid Level. The procedure is virtually identical to that described for succinylation of yeast proteins (87). In a typical experiment proteins, together with NA, were extracted from the disrupted yeast cells at pH 8.5-9.0 and centrifuged at 15,000 rpm for 30 min at 5°C. Citraconic anhydride then was added in small increments to the supernatant with constant stirring while the pH was maintained between 8.0-8.5 by adding 3.5IV NaOH. After the stabilization of the pH, the pH of the solution was decreased to 4.2 to precipitate the proteins. Protein then was separated by centrifugation, dissolved in water (pH adjusted 8.5), dialyzed extensively against water (pH 8.5) at 5°C, and lyophilized. 

Many years ago, Liss and Langen (1960a,b) showed that the most highly polymerized yeast PolyP fraction, extractable only with strong alkali (0.05 M) or when kept for a long period with diluted CaCl2 solution, is apparently firmly bound to some cell components other than RNA. The removal of RNA by RNAase had no effect on the extraction rate of this PolyP fraction. It was considered that in this case PolyP was bound to a certain protein. 

Partial hydrolysis of proteins using acid, alkali or enzymes is commonly employed to improve functionality and usefulness of novel proteins. Acid hydrolysis is the most common method for preparing hydrolysates of soy, zein, casein, yeast and gluten. Hydrolysates are used in formulated foods, soups, sauces, gravies, canned meats, and beverages as flavorants and thickeners (2,3,6). Alkaline treatments have been employed to solubilize and facilitate protein extraction from soy, single cells, and leaves. 

Ideally microbial cells should be consumable directly as food or food ingredients. However, because of their nucleic acid content the presence of undesirable physiologically active components the deleterious effects of cell wall material on protein bioavailability and the lack of requisite and discrete functional properties, rupture of cells and extraction of the protein is a necessary step. Importantly, for many food uses (particularly as a functional protein ingredient) an undenatured protein is required. For these reasons and for many potential applications of yeast protein(s) it is very desirable to separate cell wall material and RNA from the protein(s) for food applications. Much research is needed to develop a practical method for isolation of intact, undenatured yeast proteins from the yeast cell wall material to ensure the requisite nutritional and functional properties. 

A common problem associated with rupture of yeast cells and protein extraction is proteolysis. Yeast cells contain a full complement of intracellular proteolytic enzymes which may be liberated after the cells are broken either by autolysis or by mechanical disruption. These liberated proteolytic enzymes, unless inactivated during the isolation and purification of yeast proteins, hydrolyze the proteins causing poor yields of intact protein (55, 69,70). 

In conjunction with research on protein extraction from yeast, we investigated methods for the maximum recovery of protein possessing good functional properties but low in nucleic acid. Therefore, we examined the feasibility of making the yeast protein resistant to proteolysis during extraction and nucleic acid reduction. Using established extraction procedures (76), we observed... 

Figure 4. The increased quantity of protein extracted from homogenized yeast cells at pH 8.5 following succinylation...

Figure 5. The effects of succinylation during extraction of yeast protein on the endogenous ribonuclease activity (pH 6.0, 55°C)...

Solubility is a critical functional characteristic because many functional properties depend on the capacity of proteins to go into solution initially, e.g. gelation, emulsification, foam formation. Data on solubility of a protein under a variety of environmental conditions (pH, ionic strength, temperature) are useful diagnostically in providing information on prior treatment of a protein (i.e. if denaturation has occurred) and as indices of the potential applications of the protein, e.g. a protein with poor solubility is of little use in foams). Determination of solubility is the first test in evaluation of the potential functional properties of proteins and retention of solubility is a useful criterion when selecting methods for isolating and refining protein preparations (1). Several researchers have reported on the solubility of extracted microbial proteins (69,82,83,84). In many instances yeast proteins demonstrate very inferior solubility properties below pH 7.5 because of denaturation. 

Alkali treatment has been used to improve the functional properties of the insoluble protein prepared by heat precipitation of an alkaline extract of broken yeast cells (63). Heating yeast protein at pH 11.8 followed by acid precipitation (pH 4.5) yielded a preparation composed of polypeptides with increased aqueous solubility. It also increased foaming capacity of the protein 20-fold. The emulsifying capacity of the modified protein was good whereas the original insoluble protein was incapable of forming an emulsion. Alkali treatment must be carefully controlled to avoid its possible deleterious effects (24,75), e.g. alkaline treatment of yeast protein resulted in a loss (60%) of cysteine (63). 

Chemical modification of yeast protein has received limited attention though as described above it has potential as a method for facilitating recovery of yeast protein. Current studies are concerned with determination of the functional properties of proteins succinylated during the extraction. The composition of yeast proteins prepared by different methods is shown (Table 8). Noteworthy is the protein and nucleic acid concentration in the yeast isolate which differed from the concentrate in that cell wall material was removed by centrifugation. 

The absorption spectra of three yeast protein preparations prepared by different procedures were compared (Fig. 8). The presence of nucleic acid which has a X maximum at 260 nm tend to shift the absorption spectrum of yeast protein to lower wavelengths. The ratio of absorption at 280 to 260 nm is indicative of NA contamination in protein samples a ratio of more than one indicates pure protein devoid of nucleic acid whereas a ratio of 0.65 indicates approximately 30% contamination with NA. The yeast protein extracted with alkali and directly acid precipitated showed a X max at 260, a 280/260 ratio of 0.67 and contained 28%, NA determined chemically. Protein extracted in alkali, adjusted to pH 6 and incubated at 55°C for 3-5 hours, to reduce NA with endogenous ribonuclease, had a X max at 260, a 280/260 ratio of 0.8 and a NA content of 3.3% while yeast protein prepared by the succinylation procedure and precipitated at pH 4.5 showed a X max at 275 nm, a 280/260 ratio of 1.0 and nucleic acid content of 1.8. 

Legend crude protein prepared by precipitation of an alkali extract at pH 4.0 ( ) yeast protein obtained following activation of endogenous ribonuclease (82) (O), and yeast protein prepared by the succinylation procedure (O). 

To achieve success as protein ingredients for food formulation and fabrication, novel proteins should possess a range of functional properties. Frequently during extraction, refining and drying, plant and yeast proteins, intended for food uses, become denatured or altered and subsequently display poor functional properties which render them of limited use. Chemical modification provides a feasible method for improving the functional properties of plant and yeast proteins and potentially may make it possible to tailor proteins with very specific functional properties. In this review the information on modified plant proteins is reviewed and the use of succinylation for the recovery of yeast proteins with low nucleic acid is described. 

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