HIV Nef

Nef is a 205-residue myristolylated protein expressed at high levels in the early stages of HIV infection. The protein is important for the induction of AIDS and is being actively researched as a potential drug target. Unlike most HIV-1 and related proteins expressed in bacteria, Nef is recovered from the soluble fraction of E. coli extracts. The purification protocol adopted following cell breakage and low-speed centrifugation is fairly straightforward comprising two stages of ion-exchange chromatography using DEAE-Sepharose (weak exchanger) followed by Q Sepharose (strong exchanger) and finally gel filtration using Superdex 75. Characterization of the purified protein yielded the following information.

MAP30

MAP30 is a plant protein obtained from bitter melon that has anti-HIV and anti-tumor activities. The 30-kDa protein is well expressed in E. coli as a soluble protein and is purified by two stages of exchange chromatography followed by gel filtration. The clarified extract is first applied to a DEAE-Sepharose column at pH 8.0; the majority of MAP30 does not bind or weakly binds the exchange resin. The column flow-through and early eluting fractions are dialyzed against pH 6.5 buffer then fractionated using SP-Sepharose (strong cation exchanger). The final step is gel filtration using a Superdex 200 column at pH 8.0.

Human Tissue Inhibitor of Metalloprotease-2 (TIMP-2) and Hepatocyte Growth Factor isoforms (NK1 and NK2)

The TIMP families of proteins are inhibitors of the matrix metalloproteases and are critical effectors of extracellular matrix turnover. The hepatocyte growth factor (HGF) is a multifunctional protein stimulating a wide range of cellular targets. The HGF gene codes for three distinct proteins: the full-length form and two truncated isoforms that include an N-terminal domain (N) and one-kringle (NK1) or two-kringle domains (NK2). TIMP-2 (21 kDa), NK1 (21 kDa), and NK2 (30 kDa) contain multiple disulfides that stabilize the folded conformations. For example, TIMP-2, apart from having 12 cysteines that form 6 disulfides, contains a cysteine as the N-terminal residue. All three proteins were expressed in E. coli as insoluble proteins, extracted with guanidine·HCl and reductant, and the unfolded protein separated by gel filtration in a similar manner to that previously discussed. The partially purified proteins can be conveniently stored frozen in guanidine·HCl at −80°C for several years without deleterious effects on folding or recovery of active protein. The folding and oxidation of the proteins are detailed in the respective publications, Stahl et al. (1997) and Wingfield et al. (1999), but briefly, the protocols involve equilibrium dialysis incorporating urea as a co-solvent to maintain solubility during folding, and a glutathione-based oxido-shuffling system (redox buffer) to promote formation of disulfide bonds (this approach is also detailed in Basic Protocol 1 in UNIT 6.5). The final stage of the purification process is gel filtration of the folded proteins, which, apart from removing host contaminates, separates folded monomers from any misfolded and aggregated protein.

HIV-1 gp41 ectodomain

The membrane-associated glycoproteins of HIV-1 include gp120 and gp41, the latter mediating membrane fusion with the host cell. These viral envelope proteins have been the subject of intense structural analysis over the last several years as inhibition of membrane fusion, hence viral entry, is a potential drug target in the development of therapeutics for AIDS. A basic strategy in tackling membrane-associated proteins is to remove the membrane-spanning region by expressing the non-membrane-associated region or ectodomain.

Examples of protein expression and purification can be found in most biochemical journals, two which may be especially useful: Protein Expression and Purification (http://www.academicpress.com/pep), which covers advances in the expression and purification of recombinant proteins mainly from E. coli although other expression systems are often included; and Current Opinion in Biotechnology, which regularly provides updates on various aspects of recombinant protein production as well as useful reference lists. Detailed protocols are also given in the units of this Chapter and a few recent examples of protein expression and purification are discussed below to illustrate some of the general approaches used to deal with soluble and insoluble E. coli protein expression.