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Recombinant Protein Complex Expression in E. coli

William Selleck¹, Song Tan¹

¹Center for Gene Regulation, Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 5.21

DOI:

10.1002/0471140864.ps0521s52

Online Posting Date:

May, 2008

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Abstract

This unit provides procedures to design, create, and utilize polycistronic plasmids that express multicomponent protein complexes in E. coli. Both the original pST39 polycistronic expression system, which permits four genes to be coexpressed from a single plasmid, and the more recent pST44 polycistronic system, which facilitates incorporation of affinity tags and simplifies the construction of variant deletion or point mutation polycistronic plasmids, are described. Emphasis is placed on practical details for creating polycistronic expression plasmids, expressing the protein complex in E. coli, purifying the protein complex, and troubleshooting potential expression problems. Curr. Protoc. Protein Sci. 52:5.21.1-5.21.21. © 2008 by John Wiley & Sons, Inc.

Keywords: multisubunit protein complexes; polycistronic expression; affinity tags

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Introduction
Strategic Planning
Basic Protocol 1: Small-Scale Expression of Subunits and Complex in E. coli
Basic Protocol 2: Small-Scale Purification of Tagged Subunits and Complex
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Small-Scale Expression of Subunits and Complex in E. coli

Materials

Expression plasmid (see Strategic Planning)
Competent E. coli host strain such as BL21(DE3)pLysS
2×TY medium (see recipe) without antibiotics
TYE plates (see recipe) containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol
2×TY medium (see recipe) with 50 µg/ml ampicillin and 25 µg/ml chloramphenicol
1× SDS sample buffer (see recipe)
0.2 M isopropyl--thiogalactopyranoside (IPTG) in H₂O (filter through 0.22-µm filter and store up to 1 year at –20°C)
Appropriate buffer for downstream purification steps: e.g., P300-EDTA buffer (see recipe) or T100 buffer (see recipe)
Liquid nitrogen

42°C water bath
Shaking incubator (e.g., New Brunswick model C24)
Spectrophotometer to measure optical density at 600 nm
50-ml conical polypropylene centrifuge tubes (Falcon)
Tabletop centrifuge

Additional reagents and equipment for preparing E. coli cells for analysis by SDS-PAGE (unit 5.2, Support Protocol 4) and SDS-PAGE (unit 10.1)

Basic Protocol 2: Small-Scale Purification of Tagged Subunits and Complex

Materials

Affinity chromatography resin: e.g., Talon metal affinity resin (Clontech)
P300-EDTA buffer (see recipe)
Soluble cellular extract (see Basic Protocol 1, step 6)
P300-EDTA buffer containing 100 mM imidazole (see recipe)

15-ml conical polypropylene centrifuge tubes (Falcon)
Tabletop centrifuge
Sonicator: Any sonicator capable of disrupting E. coli cells will suffice, e.g., Branson S-450D 400-W probe sonicator with 1/2-in. probe)
Bio-Spin disposable spin columns (BioRad catalog #732-6008)

Additional reagents and equipment for SDS-PAGE (unit 10.1), staining of proteins in gels (unit 10.5), and immunoblotting (unit 10.10)

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Figures

Figure 5.21.1

Schematic representation of in vitro and in vivo reconstitution methods to produce a four-subunit recombinant protein complex. (A) With in vitro reconstitution, each of the four subunits is expressed and purified individually before combining to form the complex, which must be further purified from partial or misfolded complexes. (B) In contrast, in vivo reconstitution requires only a single expression step and one round of purification to produce the purified four-subunit complex.

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Figure 5.21.2

Creation of a polycistronic expression vector. The pST50Trc1-4 plasmids provide the translation cassettes for the four possible positions in the pST44 polycistronic expression vector. Specific pairs of restriction enzymes are used to subclone the translation cassettes from the pST50Trc1-4 plasmids into pST44 (XbaI-BglII for cassette 1, EcoRI-HindIII for cassette 2, SacI-KpnI for cassette 3, BspEI-MluI for cassette 4). The XbaI-BglII translation cassette from pST50Trc1 plasmid is detailed to highlight the translational enhancer () and the Shine-Dalgarno sequence (SD) that precede the coding region (black bar bracketed by NdeI and BsrGI sites). Restriction sites and coding regions for translation cassettes 1, 2, 3, 4 are shown in blue, green, yellow, and red respectively. Transcriptional promoter and termination signals on pST50Trc1-4 are present but not shown. All restriction sites shown are unique. Plasmids are not drawn to scale.

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Figure 5.21.3

Modular subcloning scheme facilitates incorporation of a wide range of affinity tag combinations. (A) Suite of affinity tags available for the pST50Trc1-4 transfer plasmids. Five N-terminal, short affinity tags are available as single noncleavable (first column), single cleavable (second column), or double cleavable tags (third column). In addition, four C-terminal, noncleavable affinity tags are provided (fourth column). These 14 affinity tag combinations are available for each of pST50Trc1, pST50Trc2, pST50Trc3, and pST50Trc4 plasmids. The names and the sequences of the six different affinity tags are provided on the right. (B) Modular subcloning scheme for subcloning genes into pST50Trc1-4 plasmids. The same BamHI-NgoMIV coding region can be subcloned into an appropriate pST50Trc1-4 plasmid to express an untagged subunit (top row), an N-terminal, noncleavable tagged subunit (second row), an N-terminal, cleavable tagged subunit (third row), or a C-terminal, noncleavable tagged subunit (bottom row). Use of this subcloning scheme adds an N-terminal Gly-Ser (from the GGATCC BamHI site) and a C-terminal Ala-Gly (from the GCCGGC NgoMIV site) to the native protein. Other subcloning schemes are also possible including NdeI-BsrGI (for untagged subunits with no non-native amino acids), BamHI-BsrGI (for N-terminal tagged subunits only), and NdeI-NgoMIV (for C-terminal tagged subunits only). The scissors icon represents the tobacco etch virus (TEV) protease recognition site or the TEV protease itself. The horizontal black bar 5¢ with respect to the NdeI site in each plasmid represents the translational initiation signals such as the translational enhancer and Shine-Dalgarno sequence.

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Figure 5.21.4

Plasmid maps for the vectors in the pST39 and pST44 polycistronic expression systems. The location of the T7 10 transcription promoter and terminator are shown for each of the plasmids, while the translational enhancer () and Shine-Dalgarno (SD) sites are shown for the transfer plasmids pET3aTr and pST50Trc1-4. The restriction sites which define the four polycistronic cassettes of pST39 and pST44 are shown in bold, as are the sites on the corresponding transfer plasmids. A specific plasmid is available for each cassette in the pST50Trc1-4 transfer plasmids. For example, pST50Trc1 would typically be used as the donor plasmid for subcloning the XbaI-BglII translation cassette into cassette 1 of the pST44 polycistronic expression vector. However, the same flanking restriction sites (shown in gray) for subcloning the translation cassette as EcoRI-HindIII (for cassette 2), SacI-KpnI (for cassette 3), and BspEI-MluI (for cassette 4) fragments are also available in pST50Trc1. This allows pST50Trc1 to function as the donor plasmid for all four cassettes, if desired. All occurrences of each of the selected restriction sites in the plasmids are shown. Priming sites for sequencing or PCR primers are depicted as labeled arrows, with their sequences shown in (C). The -lactamase gene present in each of the plasmids confers ampicillin resistance (not shown).

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Figure 5.21.5

Sample small-scale purification of a two-subunit complex. The hexahistidine (His₆)–tagged A. thaliana Gcn5 deletion construct was coexpressed with an Ada2 deletion construct. Equivalent volumes of the whole-cell extract (after sonication), extract pellet, extract supernatant, and Talon flowthrough (what did not bind to the Talon resin) are shown in lanes 1 through 4, respectively. In this experiment, 5 ml of the extract supernatant was incubated with 0.5 ml of washed Talon metal affinity resin. Note that cell lysis of this extract was incomplete given that significant amounts of all proteins were found in the pellet fraction. Since most E. coli proteins are soluble, one normally expects relatively little protein in the pellet fraction. Also note that the tagged Gcn5 and untagged Ada2 proteins bound efficiently to the Talon resin as judged by the relative absence of these proteins in the Talon flowthrough fraction. Elution using three 0.5-ml fractions of solution containing 100 mM imidazole releases the bound Ada2/Gcn5 complex, predominantly in fraction 2 (lanes 5 through 7). The asterisk marks a known E. coli contaminant, EF-Tu, found in many of our preparations of His₆-tagged protein complexes purified over Talon resin (Barrios et al., 2007). Molecular weight markers are shown in lane 8, with their molecular weights in kilodaltons shown to the right of the gel.

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Figure 5.21.6

Sample small-scale purification of a three-subunit complex. A hexahistidine (His₆)–tagged S. cerevisiae Epl1 deletion construct was coexpressed with full-length Esa1 and a Yng2 deletion construct from the same species to produce the three-subunit Piccolo NuA4 complex. Equivalent volumes of the whole-cell extract (after sonication), extract pellet, extract supernatant, and Talon flow-through (what did not bind to the Talon resin) are shown in lanes 1 through 4, respectively. In this experiment, 5 ml of the extract supernatant was incubated with 0.5 ml of Talon metal affinity resin. Efficient cell lysis occurred as evidenced by the fact that relatively little protein was present in the pellet fraction. Although less obvious than in the example in Fig. 5.21.5, the three coexpressed subunits bound efficiently to the Talon resin, as judged by the relative absence of these proteins in the Talon flowthrough fraction. Elution using three 0.5-ml fractions of solution containing 100 mM imidazole released the bound Epl1/Yng2/Esa1 Piccolo NuA4 complex, predominantly in fraction 2 (lanes 5 through 7). The asterisk marks EFTu, a contaminating E. coli protein (Barrios et al., 2007). Molecular weight markers are shown in lane 8, with their molecular weights in kilodaltons shown to the right of the gel.

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