Introduction

The pMAL-2 vectors (Fig. 1) provide a method for expressing and purifying a protein produced from a cloned gene or open reading frame. The cloned gene is inserted downstream from the malE gene of E coli, which encodes maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein (1,2) The method uses the strong "tac" promoter and the malE translation initiation signals to give high-level expression of the cloned sequences (3,4), and a one-step purification of the fusion protein using MBP's affinity for maltose (5). The vectors express the malE gene (with or without its signal sequence) fused to the lacZa gene. Restriction sites between malE and lacZa are available for inserting the coding sequence of interest. Insertion inactivates the (3-galactosidase a-fragment activity of the malE-lacZa fusion, which results in a blue to white color change on X-gal plates when the construction is transformed into an a-complementing host such as TBI (6) or JM107 (7). The vectors carry the ladq gene, which codes for the Lac repressor. This keeps expression from Ptac low in the absence of IPTG (isopropyl-P-D-thiogalacto-side) induction The pMAL-2 vectors also contain the sequence coding for the recognition site of the specific protease factor Xa (9,10), located just 5' to the polyhnker insertion sites This allows MBP to be cleaved from the protein of interest after purification. Factor Xa cleaves after its four amino acid recognition sequence, so that few or no vector-derived residues are attached to the protein of interest, depending on the site used for cloning. A purification example is shown in Fig. 2.

From Methods in Molecular Biology, vol 63 Recombinant Protein Protocols

Detection and Isolation Edited by R Tuan Humana Press Inc , Totowa, NJ

polylinker ladc

polylinker ladc

rrnB terminator

Ampr pBR on

M13 ori rrnB terminator

Ampr pBR on

M13 ori pMAL-c2, -p2 polylinker:

¡-Sac / -j malE TCG AGC TCG AAC AAC AAC AAC AAT AAC AAT AAC AAC AAC CTC GGG

|-Xmnl-1 pEcoW -| {BamH I -| j-Xba I -j [—Sa/ I —| pPs/I —j ¡-H/nd III -j

ATC GAG GGA AGG ATT TCA GAA TTC GGA TCC TCT AGA GTC GAC CTG CAG GCA AGC TTG lacZa lie Glu Gly Arg

Fig. 1. pMAL-2 vectors are shown pMAL-c2 (6646 bp) has an exact deletion of the malE signal sequence pMAL-p2 (6721 bp) includes the malE signal sequence Arrows indicate the direction of transcription Unique restriction sites are indicated

The pMAL vectors come in two versions: pMAL-c2, which lacks the N-termi-nal signal sequence normally present on MBP, and pMAL-p2 which includes the N-terminal signal sequence. Fusion proteins expressed from pMAL-c2 are expressed in the cytoplasm of Escherichia coli. These constructions generally give the highest levels of expression. Fusion proteins expressed from pMAL-p2 include a signal peptide on pre-MBP which directs fusion proteins through the cytoplasmic membrane into the periplasm. For fusion proteins that can be successfully exported, this allows folding and disulfide bond formation to take place in the periplasm of E coli, as well as allowing purification of the protein from the periplasm (8a).

To produce a fusion protein in the pMAL-2 vectors, the gene or open reading frame of interest must be inserted into the pMAL-2 vectors so that it is in the same translational reading frame as the vector's malE gene. The vectors have a polylinker containing an Xmnl site for clonmg fragments directly down-

Factor Xa cleavage site

Factor Xa cleavage site c

Grow cells

Sample 1 uninduced ceils

Add IPTG

^ Sample 2 induced cells

Divide culture and harvest cells

^ Sample 2 induced cells

Divide culture and harvest cells

Resuspend in column buffer Prepare cytoplasmic extract

Resuspend in Tns/sucrose Prepare periplasmic extract

Sample 3 crude extract Sample 4 insoluble matter

Test amylose resin binding ^

Sample 5 protein bound to amy lose

Sample 3 crude extract Sample 4 insoluble matter

Test amylose resin binding ^

Sample 5 protein bound to amy lose

Sample 6 periplasmic extract (osmotic-shock fluid)

Fig. 2 Flowchart for the pilot experiment

Sample 6 periplasmic extract (osmotic-shock fluid)

Fig. 2 Flowchart for the pilot experiment stream of the factor Xa site, as well as an EcoRl site in the same reading frame as A,gtl 1. A number of other restriction sites are also available for cloning fragments downstream of the Xmnl site or for directional cloning of a blunt/sticky-ended fragment. Inserts cloned into the Xmnl site produce a protein of interest that, after factor Xa cleavage, contains no vector-derived ammo acids (9,10). Factor Xa will not cleave fusion proteins that have a proline or arginine immediately following the arginine of the factor Xa site, so the first three bases of the insert should not code for arg or pro when cloning into the Xmnl site. If the sequence of interest was identified as a lacZ fusion m Agtl 1, it can be subcloned into the EcoRl site of the pMAL-2 polylinker directly. If the sequence is from another source, several strategies may be employed to create an appropriate fragment to subclone. It is assumed that the sequence of interest includes a translational stop codon at its 3' end; if not, one should be engineered into the cloning strategy. Alternatively, a linker containing a stop codon can be inserted into one of the downstream polylinker sites.

Section 3. contains a section on cloning, a small-scale pilot experiment to diagnose the behavior of a particular fusion protein, two methods for the affin ity purification of the fusion protein, a short method for regeneration of the amylose resin column, and methods for cleavage of the fusion protein with the specific protease factor Xa and separation of the target domain from MBP

2. Materials

In addition to the materials listed below, the standard reagents for molecular biology such as restriction enzymes and buffers, 0.5MEDTA, phenol, chloroform, T4 DNA ligase, solutions for determining protein concentration (e g., by the Bradford or Lowry method), and the materials and buffers for SDS-PAGE are required

2.1. Construction of the Fusion Plasmid

1 DNA fragment to be subcloned, preferably with a blunt end at the 5' end of the gene and a sticky end compatible with the pMAL-2 polyhnker at the 3' end

2 pMAL-c2 and/or pMAL-p2, available from New England Biolabs (Beverly, MA) (see Note 1)

3 Competent E coh TBI (or an equivalent a-complementing strain of E coli)

4 LB plates containing 100 pg/mL ampicillm, with and without 80 pg/mL X-gal

2.2. Pilot Experiment and Affinity Purification

1. Rich medium plus glucose and ampicillm (per liter) 10 g tryptone, 5 g yeast extract, 5 g NaCl, 2 g glucose, autoclave; add sterile ampicillm to 100 pg/mL

2 0 1M IPTG stock 1 41 g IPTG hemidioxane adduct (isopropyl-p-D-thio-galactoside hemidioxane adduct; mol wt 282 4) or 1 19 g IPTG (isopropyl-P-D-thiogalactoside, mol wt 238 3) Add H20 to 50 mL, filter sterilize, store at 4°C Dioxane adduct solution stable for 6 mo at 4°C, dioxane-free solution is lightsensitive, and therefore considerably less stable

3 Column buffer 20 mM Tris-HCl, 200 mMNaCl, 1 mMEDTA, pH 7 4 Optional components 1 mM sodium azide, and 10 mM p-mercaptoethanol or 1 mM DTT (see Note 2). Store at room temperature. The conditions under which MBP fusions will bind to the column are flexible, and the column buffer can be modified without adversely effecting the affinity purification. Buffers other than Tris-HCl that are compatible include MOPS, HEPES, and phosphate, at pH values around 7 MBP binds to amylose primarily by hydrogen bonding, so higher ionic strength increases its affinity Noniomc detergents such as Triton X-100 and Tween-20 have been seen to interfere with the affinity of some fusions

4 Sonicator

7 2.5 x 10 cm column

8. Centricon, Centriprep or stirred cell concentrator (Amicon), or equivalent.

9 Amylose resin, available from New England Biolabs (Beverley, MA)

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