Protein Expression and Purification

NMR structural studies require milligram quantities of isotopically labeled proteins, and the most versatile and widely used method for obtaining recombinant proteins is by expression in E. coli, since this enables a wide variety of isotopic labeling schemes to be incorporated in the NMR experimental strategy. Smaller peptides can be prepared by solid phase peptide synthesis; however, this is impractical for larger proteins and for the preparation of uniformly labeled samples, where efficient expression systems are essential. The ability to produce milligram quantities of pure proteins also facilitates functional studies that, together with structure determination, can provide important structure-activity correlations.

Some polypeptides, however, are toxic to the bacterial hosts that express them. For example, some membrane proteins and peptides, including some of bacterial origin, congest the cell membranes when they are over-expressed, and act as toxic, antibacterial agents, regardless of their actual biological functions. For these difficult polypeptides, solid-phase synthesis is not a practical alternative, because it is typically limited to sequences shorter than 50 amino acids, and while this size limit can be extended through the use of chemical ligation methods (Dawson et al. 1994; Kochendoerfer 2001) that can also be applied to membrane proteins (Kochendoerfer et al. 1999; Kochendoerfer et al. 2004), NMR studies still require bacterial expression of the polypeptide precursors for the practical introduction of various isotopic labels.

Several E. coli cell strains and expression strategies have been developed to address this problem (Miroux and Walker 1996; Rogl et al. 1998; Jones et al. 2000; Majerle et al. 2000; Opella et al. 2001; Sharon et al. 2002; Bannwarth and Schulz 2003; Booth 2003; Kiefer 2003; Lindhout et al. 2003; Smith and Walker 2003; Wiener 2004), and more recently, cell-free expression has been used to obtain milligram quantities of isotopically labeled membrane proteins (Klammt et al. 2004). Many strategies rely on the use of fusion protein tags to improve expression and facilitate purification, and many involve protein expression in inclusion bodies, to keep the hydrophobic polypeptide away from the bacterial membranes, and thus increase the level of expression. The formation of inclusion bodies also limits proteolytic degradation, and simplifies protein purification, which can be further assisted by the incorporation of an engineered His tag for metal affinity chromatography.

The TLE (a portion of the Trp ALE 1413 polypeptide) (Miozzari and Yanofsky 1978; Kleid et al. 1981; Staley and Kim 1994), and KSI (ketosteroid isomerase) (Kuliopulos et al. 1994) fusion partners promote the accumulation of expressed proteins as inclusion bodies, and have been used to express several membrane peptides and proteins ranging in size from 20 to 200 amino acids (Opella et al. 2001; Opella and Marassi 2004).

Recently, we developed a fusion protein expression vector, pBCL, that directs the expression of a target polypeptide fused to the C-terminus of a mutant form of the Bcl-2 family protein Bcl-XL, where the hydrophobic C-terminus has been deleted, and Methionine residues have been mutated to Leucine, to facilitate CNBr cleavage after a single Methionine inserted at the beginning of the target polypeptide sequence (Thai et al. 2005). As shown in Fig. 2.2, this fusion partner yields the high-level expression of membrane proteins belonging to the FXYD family of Na,K-ATPase regulators, including some that had resisted expression with TLE and KSI.

The pTLE and pBCL vectors maybe generally useful for the high-level expression of other membrane-associated proteins that are difficult to express because of their toxic properties. The use of chemical cleavage eliminates the difficulties, poor specificity and enzyme inactivation, often encountered with protease treatment of insoluble proteins. However, in cases where Met mutation is not feasible, protein cleavage from the fusion partner can be obtained enzymatically, by engineering specific protease cleavage sites for the commonly used enzymes thrombin, Fxa, enterokinase, and tobacco etch virus (TEV) protease. Thrombin and TEV retain activity in the presence of detergents, including low mM concentrations of SDS.

The pro-apoptotic Bcl-2 family protein, Bid, is activated upon cleavage by caspase-8, to release the 15kD C-terminal fragment tBid, which translocates from the cytosol to the outer mitochondrial membrane inducing massive cyto-chrome-c release and cell death (Scorrano and Korsmeyer 2003). Full-length Bid can be expressed at high levels, in E. coli, as a soluble protein, however tBid is toxic for bacterial cells. To produce milligram quantities of 15N-labeled tBid for NMR studies we used the TLE fusion protein vector (Fig. 2.2). tBid was separated from the fusion partner by means of CNBr cleavage at the engineered N-terminal Met residue, and this method yields approximately 10 mg of purified 15N-labeled tBid from 1L of culture. To avoid cleavage within the tBid segment, the four Met residues in the tBid amino acid sequence were mutated to Leu, and therefore, it was important to demonstrate that the recombinant protein retained its biological activity. Recombinant tBid, isolated from inclusion bodies, was fully active in its ability to induce cytochrome-c and SMAC release from isolated mitochondria, and retained its capacity to bind anti-apoptotic Bcl-XL through its BH3 domain despite the M97L mutation in its sequence (Gong et al. 2004).

_PLM_ tBid

BCL173 BCL99 KSI TLE TLE (vector) _ + + + +_+ (tPTG)

66.3f ill

_PLM_ tBid

BCL173 BCL99 KSI TLE TLE (vector) _ + + + +_+ (tPTG)

66.3f ill

Fig. 2.2. Expression of the membrane protein phospholemman (PLM) and of tBid with four different fusion protein plasmid vectors: pBCL173, pBCL99, pKSI,and pTLE.The gel shows total lysates from cells, transformed with each plasmid, and harvested before (-) or after (+) induction with IPTG. Fusion protein over-expression Is marked by the appearance of a distinct band at the molecular weight of the corresponding fusion protein: BCL173-PLM (29.8 kD), BCL99-PLM (21.2 kD), KSI-PLM (23.4 kD), TLE-PLM (22.1 kD), and TLE-tBId (30.0 kD) (Gong et al. 2004; Thai et al. 2005)

Fig. 2.2. Expression of the membrane protein phospholemman (PLM) and of tBid with four different fusion protein plasmid vectors: pBCL173, pBCL99, pKSI,and pTLE.The gel shows total lysates from cells, transformed with each plasmid, and harvested before (-) or after (+) induction with IPTG. Fusion protein over-expression Is marked by the appearance of a distinct band at the molecular weight of the corresponding fusion protein: BCL173-PLM (29.8 kD), BCL99-PLM (21.2 kD), KSI-PLM (23.4 kD), TLE-PLM (22.1 kD), and TLE-tBId (30.0 kD) (Gong et al. 2004; Thai et al. 2005)

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