Instrumentos para la evaluación de la solución del suelo: Lisímetros

The affinity of certain surface amino acid residues, predominately histidine, for first row transition metal ions, provides the basis for metal chelate separation o f proteins (see Section 1.3). Histidine is a relatively rare amino acid, accounting for only about 2% of the amino acids in globular proteins (Klapper, 1977) and only about half are exposed on the protein surface. If a high affinity for chelated metals is conferred on a specific protein by the incorporation o f one or more histidine residues at its C or N termini, it becomes unique and thereby easily isolated from its comtaminants. In contrast to previously discussed purification handles, metal chelating histidine tails are small and the materials for immobilised metal affinity (IMA) separation are readily available, stable and inexpensive.

A specific chelating peptide with a high affinity for immobilised metal ions can be used to purify a recombinant protein by extending the gene sequence o f the recombinant protein to code for the entire amino acids in the chelating peptide sequence. The resulting fusion protein can be purified by IMA separations. Polyhistidine extensions o f varying lengths have been utilised for immobilised metal affinity chromatography (IMAC) purifications (Lilius et al., 1991; Gentz et al., 1988; LeGrice et al., 1991). Protein expression levels may be altered by the addition o f polyhistidine tails (Skerra et al., 1991). In the production o f a polyhistidine-extended galactose dehydrogenase (Galdh-(His)$) fi"om E. coli cell extracts, the authors noted that a 2-fold increase in production could be effected by including histidine in Luna broth (Lilius et al., 1991).

Smith and coworkers (1987) were first to suggest that certain small histidine-containing peptides can be attached to proteins as purification handles for metal-affinity separations. They showed that the peptides His-Trp, His-Tyr-NH2 and His-Gly-His had unusually

high affinities for C o ^\ NP"** and Cu^^ immobilised on iminodiacetic acid (IDA)- derivatised Sephadex G-25 compared to other histidine, lysine and aspartic acid containing peptides and also that peptides with the formula His-X bound Cu^^ strongly and most bound Ni^'*’ to a lesser extent.

The high affinity of a small dipeptide could be transferred to the NH2 terminus o f a

protein that had no affinity for immobilised metal ions, thus enabling its subsequent purification. A leutenising hormone-releasing hormone (LHRH) which contains the chelating peptide, His-Trp, on the NH2 terminus had a higher affinity for a IMAC

Hochuli et al. (1988) described the affinity purification o f two polyhistidine fusion proteins containing two and six adjacent histidines, (His) 2 and (His)^, at both the C- and

N-termini of mouse dihydrofolate reductase on a nitrilotriacetic acid (NTA)- NP"*’ column. Increasing binding strength was mirrored by the increasing number o f histidine residues. The efficiency o f the polyhistidine peptide is dependent on the solvent system used during chromatography. The (His)^ tag bound more strongly than the (His) 2 tag and

elution o f the product was difficult under physiological buffering conditions but highly successful in 6 M guanidine hydrochloride (which is appropriate for the purification o f proteins which are formed in inclusion bodies). On the other hand the (His) 2 tag did not

bind in 6 M guanidine hydrochloride but worked perfectly in physiological buffers. The C-terminal chelating peptide could be removed using limited digestion with carboxypeptidase A.

A number o f HIV viral proteins have been purified by IMAC including Rev, Tat, and reverse transcriptase with a (His)g chelating peptide sequence attached to the NH2-

terminus (Cochrane et al., 1989; Gentz et al., 1989; LeGrice and Leitch, 1990). The presence o f the chelating peptide did not interfere with the biological activity o f these proteins; therefore, the fusion proteins were used directly. A single chain antibody Fv fragment contair^ a five-histidine tail at the C-terminus was purified from E. coli homogenates and periplasmic fractions on Zn^^-IDA (Skerra et al., 1991). The poly­ histidine sequence did not alter the fragment's hapten affinity, nor did it have any negative effect on the export o f the recombinant protein to the periplasm.

Ljungquist et al. (1989) synthesized a series o f histidine fusion proteins. An affinity linker Ala-His-Gly-His-Arg-Pro was polymerised at gene level to two, four and eight copies and fused to the 3' end o f a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end o f a gene encoding P-galactosidase. Fusion proteins o f both types, with zero or two copies o f the linker showed little or no binding to immobilised Z n ^\ while a relatively strong interaction was observed for the fusions based on four or eight copies o f the linker. This suggest that the Zn^"*" interaction o f histidines is relatively weak and that multivalent binding is needed to achieve efficient recovery o f the recombinant protein. Zn^+ has been shown to have a low binding affinity for native proteins (Sulkowski, 1985). This makes the system suitable for applications in which low background binding o f non-recombinant proteins is desired.

Angiotensin 1, a decapeptide containing a His-X2-His sequence was used as an affinity

tail for a one-step IMAC purification on Zn^+-IDA columns (Beitle and Attai, 1993). This peptide was shown to exhibit strong metal binding properties (Sulkowski, 1982;

Hutchens and Yip, 1990; Yip et al., 1989). In general, proximal metal-binding residues are necessary for a protein to display an affinity toward Zn^+-IDA (Hemden et al., 1989).

The composition and conformation o f a protein, in particular the proximity and surface exposure o f histidine residues, dictate the affinity a protein may have toward a given metal chelated to a stationary phase (Sulkowski, 1985). Both Suh et al. (1991) and Todd et al. (1991) engineered synthetic metal binding sites consisting o f two solvent exposed histidines separated by a single turn o f an a-helix (His-X^-His variants) on to the surface, and the N-terminus respectively, o f a protein. In this orientation the e nitrogens o f the imidazole groups from both histidines can co-ordinate a single metal. This motif forms part o f the metal co-ordination sites in a number o f metalloproteins, including 'zinc- finger' proteins (Berg, 1988; Parraga et al., 1988) and thermolysin (Holmes and Mathews, 1982). The affinities of the His-Xg-His sites for Cu^"^ dramatically increased the proteins' retention on a metal affinity column compared to proteins containing independent histidines. Incorporation of the His-X^-His site into a potentially flexible N- terminal region yielded a synthetic metal binding protein whose affinity for metal ions was sensitive to environmental conditions which affect helix structure or stability. The afiSnity o f the synthetic metal-binding cytochrome c for Cu^^-IDA-PEG (Todd et al., 1991) increased with pH fi'om 5.5 to 8.0. However above pH 8.0 the copper binding affinity o f the His-X^-His cytochrome c decreased rapidly. The helicity o f short peptides in solution depends on the temperature, pH, ionic strength and replacements in the amino acid sequence (Finkelstein et al., 1991).

The use o f immobilised metal affinity separation with chelating fusion proteins has several advantages. The technique can handle either soluble or insoluble recombinant proteins. Supernatants o f lysed cells can be applied directly to an IMA system column with or without using salt concentrations. Dénaturants, such as detergents, urea, guanidine hydrochloride, and organic solvents, can be used in the IMAC chromatographic separation when the expression product is insoluble. The bound proteins can be eluted with a pH gradient or with increasing concentrations o f a displacing ligand, such as imidazole, in cases in which the protein of interest cannot tolerate a low pH. IMA supports are also resistant to microbial contamination because o f the high metal content. One of the major advantages o f chelating fusion proteins peptides is their small size. The efficiency of expression is optimal because the purification handle is tiny compared to that o f most fusion proteins whose purification handle is large or larger than the protein o f interest. Another advantage of small size means that affinity purification can be achieved without interference with the biological activity o f the

protein which then eliminates the need to remove the chelating peptide (except for in vivo use).