Protein Production in Saccharomyces cerevisiae for Systems Biology Studies

C H A P T E R

E L E V E N

Protein Production in Saccharomyces cerevisiae for Systems Biology Studies Naglis Malys,*,† Jill A. Wishart,*,‡ Stephen G. Oliver,*,‡,1 and John E. G. McCarthy*,†,§,2 Contents 1. 2. 3. 4.

Introduction Comparison of Commonly Used Expression Systems Comprehensive Libraries for Protein Production in S. cerevisiae Protocols for Protein Expression and Purification from Tagged Collections of S. cerevisiae 4.1. Cell growth and protein expression 4.2. Cell disruption 4.3. Protein purification 5. Protein Analysis and Quantification 6. Protein Use in Proteomics and Enzyme Kinetics Measurements 7. Concluding Remarks Acknowledgments References

198 199 199 202 203 205 205 207 209 209 209 210

Abstract Proteins together with metabolites, nucleic acids, lipids, and other intracellular molecules form biological systems that involve networks of functional and physical interactions. To understand these interactions and the many other characteristics of proteins in the context of biochemical networks and systems * Manchester Centre for Integrative Systems Biology, The University of Manchester, Manchester, United Kingdom Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, United Kingdom { Faculty of Life Sciences, Michael Smith Building, The University of Manchester, Manchester, United Kingdom } School of Chemical and Analytical Engineering, The University of Manchester, Manchester, United Kingdom 1 Present address: Cambridge Systems Biology Centre, Department of Biochemistry, Sanger Building, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom 2 Present address: School of Life Sciences, Gibbet Hill Campus, University of Warwick, Coventry, United Kingdom {

Methods in Enzymology, Volume 500 ISSN 0076-6879, DOI: 10.1016/B978-0-12-385118-5.00011-6

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2011 Elsevier Inc. All rights reserved.

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biology, research aimed at studying medium and large sets of proteins is required. This either involves an investigation focused on individual protein activities in the mixture (e.g., cell extracts) or a protein characterization in the isolated form. This chapter provides an overview on the currently available resources and strategies for isolation of proteins from Saccharomyces cerevisiae. The use of standardized gene expression systems is discussed, and protein production protocols applied to the data generation pipeline for systems biology are described in detail.

1. Introduction The necessity for producing comprehensive collections of active proteins is becoming critically important as systematic studies of biological processes and attempts at reconstituting various biological systems are under way. This requires accelerated speed and high efficiency from the protein production pipeline. In the past few decades, overexpression in Escherichia coli has established itself as a mainstream strategy for efficient protein production (Gold, 1990; Junge et al., 2008; Makrides, 1996; Tabor and Richardson, 1985; Zerbs et al., 2009). Yeast, baculovirus-insect, mammalian, and other bacterial or cell-free expression systems have also been studied and used effectively (Cregg et al., 2009; Jarvis, 2009; Junge et al., 2008; Katzen et al., 2005; Malys and McCarthy, 2011; Zerbs et al., 2009). However, the most fundamentally studied eukaryote—Saccharomyces cerevisiae—has attracted less attention as a possible tool for protein production. This is due to the following problems: (1) difficulties in disrupting the thick cell wall to successfully extract intracellular proteins; (2) absence of very strong transcriptional and translational systems, which could offer very high protein synthesis levels similar to those available in E. coli. Despite the shortcomings, in the past decade, S. cerevisiae has become a reliable source for protein production and its advantages (such as good genetic characterization, ease, and safety of the organism to work with, relatively inexpensive protein production process, tightly regulated and efficient expression systems, appropriate protein folding, and some level of posttranslational modification) have been better appreciated. This chapter reviews commonly used protein production systems for yeast and describes in more detail the comprehensive gene-expression libraries that can be used for the production of proteins in a research environment focused on systems biology. Specific procedures for cell preparation, gene expression, and protein purification and analysis form a major part of this chapter.

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2. Comparison of Commonly Used Expression Systems Choosing an appropriate host for expressing proteins of interest is a decisive step for any biological research project. Selection of an inadequate expression system can result in several undesirable consequences for the protein: poorly expressed, misfolded, or lacking natural posttranslational modifications. A comparison of commonly used expression systems highlighting their positive and negative characteristics is shown in Table 11.1. For studies that require characterization of whole biological systems and, hence, a relatively large number of proteins, other important factors to consider are speed, reproducibility, and the ability to express and purify all required proteins using a single (or a few very similar) expression system(s). The intended use of the produced proteins is also important in decision making, as different quantities and qualities of the protein sample might be required for in vitro activity assays or structural studies, for example.

3. Comprehensive Libraries for Protein Production in S. cerevisiae For protein production in S. cerevisiae, a number of comprehensive gene-overexpression libraries have been created by inserting yeast ORFs into vectors in which gene expression is usually controlled by the strong galactose-inducible GAL1 promoter. Of the currently available libraries, two collections stand out as the most complete and suitable for yeast protein overproduction (Boone et al., 2007). First, the Yeast ORF Collection has been developed by collaborative work between Eric Phizicky’s and Mike Snyder’s groups (Gelperin et al., 2005). In this collection, yeast ORFs are placed under the control of the inducible GAL1 promoter and cloned into individual plasmid vectors designed for tagged protein overexpression. A C-terminal 19-kDa tandem fusion tag is formed of 6xHis and hemagglutination (HA) protein domains, a 3
C protease cleavage site, and two IgG-binding domains from Staphylococcus aureus protein A. This tag configuration enables robust affinity purification, sensitive immunodetection, and partial tag removal (i.e., protein A). Over 4900 S. cerevisiae genes can be expressed and proteins purified by using this collection (Gelperin et al., 2005). The Yeast ORF Collection has been used successfully for the identification and characterization of enzymatic activities ( Jackman et al., 2007, 2008; King et al., 2009) and protein interaction studies (Kung and Snyder, 2006; Li et al., 2008).

Table 11.1 Characteristics of commonly used expression systems for protein production Expression system Characteristics

Expression level

Bacteria

Moderate–high Usually several milligrams of protein per liter of culture, gram per liter can be achieved in some instances Time efficiency High Cell growth 20–30 min/ division Time required for 3–4 days protein production Cost of resources Low Process operation Easy and scalability Density of biomass High Recombinant Low protein quality

Yeast

Insect

Mammalian

Plant

Cell free

Low–high Low Low–moderate Moderate–high Low–high Broad range, Occasionally, up Several Occasionally, up Rarely, up to depending on micrograms to few several to several cell type used of protein milligrams of milligrams of milligrams of for extract per gram of protein per liter protein per liter protein per preparation cell tissue of culture of culture liter of culture

High 80–120 min/ division 5–7 days

Low Low 18–24 h/division 24 h/division 2–4 weeks

3–8 weeks

Low 18–24 h/ division 3–8 weeks

Low Moderate

High Difficult

High Difficult

Low Difficult

Moderate–high Moderate

Low High

Low High

Low–high High

Very high N/A less than 1 day

High Easy and expensive N/A Low–moderate

Folding Glycosylation

Frequently improper No

Phosphorylation, No acetylation, and acylation No Gammacarboxylation Development and Very high reliability level of expression system

Rarely improper Usually proper

Usually proper

Usually proper Depends on extract source Yes Depends on extract source Yes Depends on extract source

O-linked

O-linked

Yes

Yes

Yes

Yes

No

Yes

Yes

–

High

Moderate

Moderate

Low

High

Information from the following sources: Brondyk, 2009; Junge et al., 2008; Lindbo, 2007; Sabate et al., 2010; Yin et al., 2007; www.invitrogen.com.

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The second major resource is the Yeast GST-Tagged Collection, which has been assembled by the groups of Charles Boone and Brenda Andrews (Sopko et al., 2006) based on the previously developed overexpression plasmid library (Zhu et al., 2001). It includes more than 5000 strains that allow the overexpression of more than 80% of S. cerevisiae proteins. In each plasmid, the yeast ORF is under the control of the inducible GAL1/10 promoter and contains GST-6xHis tag at the N-terminus. This allows reliable protein overexpression under galactose induction, affinity purification (using glutathione sepharose), and immunodetection (by anti-GST antibodies). GST-tagged protein screens have been used in phenotype and pathway mapping (Sopko et al., 2006), transcription factor functional analysis (Chua et al., 2006), and many other applications. It has also been proposed as a metabolite screening tool for systems biology (Kell, 2004). An additional comprehensive library, which expresses proteins at endogenous levels, is most useful for protein complex purification. The Yeast TAP-Tagged Collection was constructed by the O’Shea and Weissman groups (Ghaemmaghami et al., 2003) and uses the tandem affinity purification (TAP) strategy (Puig et al., 2001; Rigaut et al., 1999). In each strain of this collection, a C-terminal-tagged ORF is under the control of the endogenous promoter at its original chromosomal location. The TAP tag includes a calmodulin-binding peptide, TEV cleavage site, and the IgG-binding domains of protein A (which, similarly to the Yeast ORF Collection, allows affinity purification, immunodetection, and partial tag removal). The Yeast TAP-Tagged Collection has been used for global quantitative analysis of the yeast proteome (Ghaemmaghami et al., 2003). Over 4200 proteins were detected, and absolute protein levels in exponentially growing cells were quantified using Western blot analysis. C-terminal and N-terminal TAP tag strategies have also been used successfully for the identification, purification, and functional characterization of distinct protein complexes (Fatica et al., 2002; Herna´ndez et al., 2006; Malys and McCarthy, 2006; Malys et al., 2004; Panigrahi et al., 2007) and in highthroughput functional analyses (Gavin et al., 2006; Krogan et al., 2006).

4. Protocols for Protein Expression and Purification from Tagged Collections of S. cerevisiae In order to overexpress and purify proteins for systems biology studies, the Yeast ORF Collection (Gelperin et al., 2005) can be used as the primary source. This section describes procedures that have been used to produce a comprehensive collection of enzymes for studying individual biochemical networks and generating data for mathematical modeling. To produce

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enzyme kinetics parameters, proteins need to be isolated in submicrogram quantities with their full enzymatic activity retained. For example, for a systems analysis of the glycolysis and trehalose biosynthesis pathways, 35 proteins were initially selected as core components of the networks. Thirtytwo proteins were identified to be available for expression from the Yeast ORF Collection and, after initial expression and a first round of binding to IgG sepharose (Fig. 11.1), 26 proteins were expressed in quantities sufficient for application in systems biology studies. Table 11.2 lists the successfully purified enzymes and the quantities obtained from single-culture experiments. In order to produce required proteins that are not available from the Yeast ORF Collection, the Yeast GST-Tagged Collection and Yeast TAP-Tagged Collection can be used as alternatives. The ability to coexpress protein subunits from the Yeast TAP-Tagged Collection under control of their native promoters and with natural stoichiometries allows the purification of enzyme complexes formed of different peptides (e.g., heterooctamer of Pfk1/Pfk2 for phosphofructokinase).

4.1. Cell growth and protein expression All three S. cerevisiae strain collections (Yeast ORF, Yeast GST-Tagged, and Yeast TAP-Tagged) are available from Open Biosystems (www. openbiosystems.com). Normally, strains are stored at  80
C in 96-well plates. Yeast ORF and Yeast GST-Tagged libraries include expression vectors pBG1805 and pEGH. Both contain the URA3 gene, which is required by S. cerevisiae strains Y258 (MATa, pep4-3, his4-580, ura3-52, leu2-3, 112) B Tps1

Trehalose

Pgm2 Ugp1 Pgm1 Nth1 Ath1 Tps3 Tps2 Nth2 Tsl1

Glycolysis

Fba1 Pdc1 Hxk1 Glk1 Gpm1 Pyk2 Adh2 Tpi1 Hxk2 Pfk1 Pdc5 Pdc6 Eno2 Adh3 Pfk2 Pgi1 Cdc19 Eno1 Tdh1 Pgk1 Tdh2 Adh1 Pgm2 Ugp1 Pgm1 Nth1 Ath1 Tps3 Tps2 Nth2 Tsl1 Tps1

A

Figure 11.1 Proteins expressed in strains from the Yeast ORF Collection. SDS/PAGE analysis of IgG sepharose-bound enzymes from glycolysis and trehalose biosynthesis pathways (A) and Western blot analysis of trehalose biosynthesis enzymes (B).

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Table 11.2 Examples of purified enzymes with quantities and percentages of full-length proteins in sample preparation

Protein

Percentage of full-length Quantity, Protein mg/culturea protein

Percentage Quantity, of full-length mg/culturea protein

Adh1 Cdc19 Eno1 Eno2 Fba1 Glk1 Gpm1 Hxk1 Hxk2 Pdc1 Pdc5 Pdc6 Pfk1

0.08 0.48 0.56 1.62 0.38 0.70 1.02 0.54 1.56 0.48 0.63 0.39 0.25

0.12 0.08 0.35 0.11 0.16 0.32 0.80 0.08 0.24 0.16 0.28 0.64 0.90

97 96 93 98 85 98 99 99 97 97 96 94 88

Pfk2 Pgi1 Pgk1 Tdh1 Tdh3 Tpi1 Tps1 Tsl1 Tps2 Nth1 Pgm1 Ugp1 Pgm2

97 97 97 95 93 95 95 82 97 98 98 97 99

The majority of proteins were purified from the Yeast ORF Collection. Gpm1 and Eno2 were from the Yeast GST-Tagged Collection. Cdc19, Tpi1, and Tdh3 were from the Yeast TAP-Tagged Collection. a Represents average calculated from values determined by two independent protein quantification techniques as described in Section 5.

and BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0), respectively, when strains are cultured in a synthetic medium without uracil (S-Ura medium: 1.7 g/L YNB (w/o) amino acids and (w/o) ammonium sulfate, 5 g/L ammonium sulfate, 20 mg/L adenine, 20 mg/L arginine, 30 mg/L isoleucine, 20 mg/L histidine, 60 mg/L leucine, 30 mg/L lysine, 20 mg/L methionine, 50 mg/L phenylalanine, 20 mg/L tryptophan, 30 mg/L tyrosine, and 150 mg/L valine) to select for the expression plasmid containing the ORF of interest. Strains from the Yeast TAP-Tagged Collection (constructed in the haploid BY4741 genetic background) can be grown in synthetic complete medium without histidine, or in rich YPD medium. To start with, 96-well stock plates are thawed. The foil seals of the wells containing the strains of interest are pierced, and cells are grown by pipetting 10 mL of cell culture and spreading it for single colonies on S-URA/ raffinose agar plates (Yeast ORF and Yeast GST-Tagged Collections) or YPD agar plates (Yeast TAP-Tagged Collection). Cells are grown on agar plates at 30
C for 3–5 days. Single colonies are then used to inoculate liquid medium, cultures are first grown overnight at 30
C, 220 rpm in 5-ml volumes of S-Ura with 2% (w/v) raffinose in 25-mL flasks for Yeast ORF and Yeast GST-Tagged Collections, or 50 ml of YPD in 300-mL flasks for Yeast TAP-Tagged Collection. Overnight cultures of strains from the Yeast

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TAP-Tagged Collection are diluted into 800-mL YPD in 3- L flasks and grown for 6–8 h to OD600 2–4, followed by cell harvesting and then treated as described later in this section. Overnight cultures of Yeast ORF and Yeast GST-Tagged Collections are diluted into 60-mL S-Ura with 2% (w/v) raffinose in 300-mL flasks and grown for 24 h at 30
C and 220 rpm, followed by dilution into 760-mL S-Ura/2% raffinose to OD600 0.05 in 3-L flasks and overnight growth to OD600 0.6–0.8. ORF expression is induced by adding 40 mL of 40% (w/v) galactose to each culture bringing the total volume to 800 mL and allowing cells to grow for 5 h. All cultures are harvested by centrifugation at 6000g and washed twice with 50 mL of ice-cold water. Protein purification procedures can be initiated immediately, or cell pellets can be stored at 80
C. Occasionally, to produce a minimum of 1 mg protein, the volume of culture has to be adjusted by increasing the number of flasks for each strain of interest.

4.2. Cell disruption Cells from each 800 mL culture are resuspended in 4 mL of disruption buffer (10 mM Tris–Cl, pH 8.0, 150 mM NaCl, 2.5 mM PMSF, 2.5 mg/mL leupeptin, 2.5 mg/mL pepstatin, and 1 Protease inhibitor cocktail tablet (Roche)) and are lyzed mechanically by bead beating in a Mini-BeadBeater16 (Biospec Inc.). Standard 2 mL screw-cap microcentrifuge tubes containing 0.4–0.5 mL of 0.425–0.600 mm glass beads (Sigma) and 0.8–1.0 mL of cell suspension in disruption buffer are subjected to four cycles of shaking (3450 oscillations/min) for 30 s, each with a 1-min interval on ice between each cycle. Then, tubes are centrifuged at 8000g for 5 min and the supernatant is collected. A new aliquot of disruption buffer is added to the cell/debris pellet, bead beating is repeated, and the supernatant collected as above. Lysis efficiency can be checked microscopically by determining the ratio of the cell debris to intact cells. When the bead-beaten cell suspension is centrifuged, the pellet usually contains two layers: a top lighter layer of cell debris and organelles from disrupted cells, and a bottom darker layer of intact cells. The ratio between the top and bottom layers of 5:1 or higher is a good indicator of efficient cell lysis. The combined cell lysate from both supernatants from the above centrifugations is subjected to a further centrifugation step at 20,000g for 15 min, and the resulting supernatant is used for the protein purification.

4.3. Protein purification Immediately after proteins are extracted from yeast cells, soluble fractions are applied to the protein purification procedure. First, affinity purification on IgG sepharose is performed for Yeast ORF and Yeast TAP-Tagged Collections, or glutathione sepharose purification is used for Yeast GST-Tagged Collection.

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Extracts (10–12 mL) in disruption buffer are combined with 100–120 mL of 10% NP40 detergent and added to 200–400 mL of IgG Sepharose beads (GE Healthcare) for Yeast ORF and Yeast TAP-Tagged Collections, and to 200–400 mL of glutathione sepharose beads for Yeast GST-Tagged Collection, which have both been prewashed three times with 2 mL of washing buffer (10 mM Tris–Cl, pH 8.0, 150 mM NaCl, 0.1% NP40). The suspension is mixed in a 15-mL tube at 4
C for 2 h and then passed through a 9cm high Poly-Prep column (Bio-Rad). Unbound proteins are separated by gravity from beads with bound protein of interest. After unbound proteins are removed, both types of beads are subsequently washed six to eight times with 10 mL of washing buffer, twice with 5 mL of protease cleavage buffer (10 mM Tris–Cl, pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.1% NP40; for Yeast ORF and Yeast TAP-Tagged Collections only), and transferred to 2-mL microcentrifuge tubes. IgG Sepharose beads are resuspended in 400 mL of protease cleavage buffer and with 0.5–2 units of PreScission protease (human rhinovirus 3
C protease–GST fusion, GE healthcare) for the Yeast ORF Collection, or with 2–10 units of AcTEV Protease (Tobacco Etch Virus (TEV) protease, Invitrogen) for Yeast GST-Tagged Collection, and vigorously mixed (600 rpm) in thermomixer (Eppendorf) for 16 h at 4
C. Then, the PreScission protease can be removed by additional incubation with 20 mL of pre-equilibrated glutathione sepharose 4B beads (GE healthcare) for 1 h. For the Yeast GST-Tagged Collection, glutathione sepharose beads are resuspended in 400 mL of elution buffer (50mM Tris–Cl, pH 7.5; 150 mM NaCl; 10 mM Glutathione, reduced form) and vigorously mixed (600 rpm) in thermomixer for 1 h at 4
C. To collect proteins of interest, which have been released from IgG Sepharose by the protease digestion, or from glutathione sepharose by the reduced form of glutathione, the 2-mL tubes with bead suspensions are centrifuged at 300g for 1 min and supernatants with proteins of interest are collected. The beads are washed twice more with 300 mL of either protease cleavage buffer or elution buffer. The resulting 1 mL supernatant, containing the protein of interest, is clarified by passage through a SPIN-X filter centrifuge tube, 0.22 mm cellulose acetate (Corning, Inc.). The buffer in the purified protein sample can be exchanged for any desired storage buffer using Vivaspin 2 centrifugal filters (Sartorius). For enzyme kinetics studies in our systems biology investigations, proteins are stored in 50 mM MES, pH 6.5, 150 mM KCl, 2 mM MgCl2 at 4
C or 80
C. As shown in Fig. 11.2, the success of each step of purification of an individual protein can be assessed by analyzing intermediates and the final protein sample on SDS–PAGE (Laemmli, 1970). In the case of the Yeast ORF Collection, where the 6xHis motif protein tag remains, the protein can be subjected to further purification using Ni2þ affinity chromatography (e.g., Ni-NTA Superflow; Qiagen). If required,

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1

2

3

4

5

6

7

8

9

Figure 11.2 SDS–PAGE representing key stages of protein purification from S. cerevisiae: enolase isoenzymes Eno1 and Eno2. Lane 1 represents a protein molecular weight marker containing 116, 66, 45, 25, and 18 kDa protein standards. Lanes 2, 4, 6, 8 are for Eno1; lanes 3, 5, 7, 9 are for Eno2. Protein bound to the IgG sepharose (2 and 3), cleaved with 3
C protease (4 and 5), eluted from the IgG sepharose (6 and 7), and after purification on HisTrap HP and HiTrap desalting columns (GE Healthcare).

1–2 mM MgCl2 and 10% (w/v) glycerol can be used in disruption, washing, elution, and storage buffers. For unstable and environment-sensitive enzymes, it is advisable that proteins are tested for different purification and storage conditions and assayed immediately after purification is complete.

5. Protein Analysis and Quantification Quantities and concentrations of purified enzymes are determined TM using protein QuantiPro BCA Assay Kit (Sigma–Aldrich; Smith et al., 1985). The quantity of protein of interest and quality of sample preparation can be further evaluated using 2100 Bioanalyzer (Agilent Technologies) as shown for neutral trehalase (Fig. 11.3). Proteins are separated using gel electrophoresis principles and microfluidic capillary technology, which are replicated into the chip format. During separation, proteins are detected by laser-induced fluorescence. The manufacturer’s protocol and Protein 230 kit are employed for sample preparation and chip priming. Agilent Expert Software is used for data analysis. Table 11.2 lists some successfully purified enzymes, their quantities, and the percentage yield of the full-length protein of interest. It should be noted that some proteins are unstable or susceptible to proteases resulting in lower percentage yields of full-length proteins in the protein preparation (e.g., Tsl1).

A

[kDa]

[kDa] NTH1

Ladder

NTH1 1/4

NTH1

NTH1 1/2

NTH1 1/4

BSA 3 mg/mL

BSA 2 mg/mL

BSA 1 mg/mL

BSA 0.5 mg/mL

240

240

150

150

95

95

63

63

46

46

28

28

15

15

4.5

4.5

1

L

2

3

4

5

6

7

8

9

[FU]

10

00

NTH1 0.

B

NTH1 1/2

250

64

2.

59

200

150

0.

00

0.

00

100

0 .0

.5

60

86

60

43

.6

.4

8

11 0

1.

7

07

50

0

4.5

C

Size [kDa] 4.5 7.1 9.3 25.9 54.5 61.8 85.2 91.8 240

15

Rel. Conc. [ng/ml] 0 0 0 43.5 60.6 101.1 86.6 642.6 60

28

46

63

Calib. Conc. [ng/ml] 0 0 0 0 0 0 0 674.4 0

95

150

Time corrected area 750.9 163.5 219.5 13.7 19.1 31.9 27.3 202.8 18.9

240

% Total 0 0 0 4.7 6.5 10.8 9.3 68.7 0

[kDa]

Observations Lower marker System peak System peak

Calibrated protein Upper marker

Area 136.7 30.9 43.5 3.3 5.4 9.4 8.6 64.7 8.1

Figure 11.3 Protein quantification and quality of sample preparation evaluated using 2100 Bioanalyzer. (A) Gel-like image of a 14–230 kDa chip run of partially purified neutral trehalase (Nth1) from S. cerevisiae and bovine serum albumin (BSA) standard. Protein samples were loaded on the protein chip using Protein 230 kit in the following order: molecular weight ladder (4.5–240 kDa), two repeats of various dilutions (undiluted, two and four times) of Nth1 protein after the first step of purification on IgG Sepharose column, last four lanes are different concentrations of BSA (3, 2, 1, and 0.5 mg/mL). (B) Electropherogram shows chip analysis of the partially purified enzyme (undiluted Nth1) sample that contains some truncated protein fragments. (C) Peak table represents quantitative characteristics of the contents in the first sample (undiluted Nth1). Nth1 was quantified on the basis of internal and BSA standards. Based on the relative quantification with the Bioanalyzer, the overall concentration for full-length Nth1 was estimated to 642.6–674.430 ng/mL accounting for 68.7% of the total protein in the sample.

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6. Protein Use in Proteomics and Enzyme Kinetics Measurements Systems biology requires experimental data that are quantitative, comprehensive, and compatible with mathematical/computational simulation of a biological system (Kell, 2006). This essentially entails establishing: (1) in vivo levels of biological molecules (e.g., proteins and metabolites); (2) molecular changes and dynamics (e.g., enzymatic reactions); and (3) spatial distribution of where such changes occur (e.g., in the cellular cytoplasm). Protein production contributes to the collection of these data in at least two areas. First, purified proteins can be used as internal standards for quantification and quality control in establishing protein levels of the biological system (Carroll et al., 2011). Second, proteins play key roles in measuring molecular interactions and changes including signaling, transport, and enzymatic reactions (Franklin and Ullu, 2010; George et al., 2009; Herna´ndez et al., 2006; Malys et al., 2002, 2009; Messiha et al., 2011; Norris and Malys, 2011; Smallbone et al., 2011).

7. Concluding Remarks Overexpression of tagged proteins brings enormous advantages: it significantly increases solubility and throughput allowing purification of multiple proteins of choice (Malhotra, 2009). It also brings several limitations and problems such as functional and structural issues. Occasionally, the position of affinity tags on the protein can interfere with the folding, complex formation, and functionality of the protein. It is sensible to employ, in parallel, different types of constructs with both C-terminal and N-terminal tags. If the functionality of one form of the tagged protein is reduced or lost, due to the position of the tag, the other form of tagged protein can potentially be used thereby reducing chances of a negative impact of the tag. Combined use of Yeast ORF and Yeast GSTTagged Collections (Gelperin et al., 2005; Sopko et al., 2006) provides this complementary approach for the purification of approximately 80% of all S. cerevisiae proteins.

ACKNOWLEDGMENTS We acknowledge the support of the BBSRC/EPSRC grant BB/C008219/1 “The Manchester Centre for Integrative Systems Biology (MCISB).” We thank Douglas B. Kell for his early contribution to this work.

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