[5 kDa

Figure 1. Schematic presentation of staphylococcal protein A (SpA) and streptococcal protein G (SpG). (a) The complete SpA gene product and the 7-kDa B-domain analog Z, represented in the most widely used divalent form. (b) The SpG gene product, with the separate albumin- and immunoglobulin-binding regions indicated, and a presentation of some of the most commonly used albumin-binding affinity tags. BB (25 kDa) was the first isolated serum albumin-binding affinity tag (14). More recently, the new tags ABP (15 kDa) and ABD (5 kDa), containing two or one serum albumin-binding motifs, respectively, were designed on the basis of postulated borders for the albumin-binding protein domains (13,47). The three postulated minimal albumin-binding motifs are indicated as bars above the SpG.

of soluble fusion proteins to very high concentrations within the Escherichia coli cell (42,43).

To create a "second-generation" affinity tag, an engineered IgG-binding domain, Z, was designed based on domain B of SpA (Fig. 1a) (12). This domain naturally lacks methionines, making it resistant to CNBr cleavage. In addition, an NG dipeptide sequence present in all native domains was changed to NA by altering the glycine codon to a codon for alanine, rendering the Z-domain also resistant to cleavage by hydroxylamine (12). Analysis of the interaction between IgG and Z domains, polymerized to different multiplicities, demonstrated that a dimeric form, ZZ (Fig. 1a), was the optimal fusion partner by its strong IgG-binding (44) and efficient secretion (12).

A different affinity-tag system, which has a number of features in common with the SpA system but also some additional advantageous properties, is based on the serum albumin-binding region of streptococcal protein G (SpG; Fig. 1b) (14,45). The regions of SpG mediating binding activities to serum albumin and IgG have been shown to be structurally separated (Fig. 1b) (14). Figure 1b illustrates SpG schematically and presents some existing variants of albumin-binding affinity tags. SpG binds to serum albumins of different mammalian species, including humans, mouse, and rat (45). The complete region comprises three serum albumin-binding motifs (48), each being approximately 48 amino acids in size. One of these postulated minimal motifs has been produced and tested for its serum albumin binding (47) and is considered to be a suitable affinity tag in a mono- or divalent fashion. However, tags comprising one, two, two-and-a-half, or three serum albumin-binding motifs (Fig. 1b) have all been successfully produced and used in different applications (4,13,27,4750). The affinity tags, denoted BB, ABP (for albumin-binding protein), and ABD (for albumin-binding domain) (Fig. 1b), are proteolytically stable, highly soluble, and possible to produce at high yields (13,28,48,51). The 48-amino acid albumin-binding domain (ABD) was recently subjected to a heteronuclear nuclear magnetic resonance (NMR) analysis, concluding that it constitutes a three-helix bundle (52), remarkably similar to that of the IgG-binding domains of SpA.

Host Cell Systems, Affinity Resins, and Detection Systems

To date, a multitude of proteins have been produced as fusions to the IgG-binding domains of SpA (native domains or the engineered Z-domains; see following), in host cells from all five kingdoms of living organisms: (1) Gramnegative bacteria, such as E. coli (more than 100 published examples, including Refs. 13,28,53-55) and Salmonella ty-phimurium (58,57), as well as Gram-positive bacteria such as Staphylococcus aureus (58,59), Streptococcus sanguis (80), and Bacillus subtilis (81); (2) the yeast Saccharomyces cerevisiae (82-88); (3) plants (87); (4) insect cells (88,89); and (5) mammalian cells (70-75).

The SpG-derived expression systems have found several applications (as are described later), some completely new, and some as a complement to the SpA/ZZ systems. The albumin-binding tags have not yet been evaluated for production in all host organisms but have been success fully expressed for different purposes in Gram-negative bacteria such as E. coli (13,14,28,47,51), Gram-positive bacteria such as Staphylococcus xylosus and Staphylococcus carnosus (48,78-80), and mammalian cells such as Chinese hamster ovary (CHO) cells (81).

The SpA fusion proteins, which bind to the Fc region of IgG, are commonly affinity purified using IgG-Sepharose (50,82) and the serum albumin-binding SpG fusions are normally purified on human serum albumin (HSA) Sepharose (14,50). However, other matrices, such as protein-coated paramagnetic beads, have also been utilized (83). SpA (ZZ) and SpG (BB, ABP, or ABD) fusion proteins can be easily detected during expression, without the need of specific antisera, by blotting techniques after poly-acrylamide gel electrophoresis (PAGE) by taking advantage of their IgG- (Fc) and HSA-binding properties, respectively. SpA fusions are efficiently stained using a commercially available complex of rabbit anti-horseradish peroxidase-IgG and horseradish peroxidase (13). SpG fusions are stained using biotinylated HSA and conjugated streptavidin-alkaline phosphatase (13).

Expression Vector Systems for E. coli

A number of expression vectors based on gene fusions to the SpA gene, often in the form of the divalent SpA analogue ZZ (12), or all five native IgG-binding domains, have been developed for recombinant protein production in E. coli (11,82,84). The promoter and secretion signals of SpA are functional also in the Gram-negative E. coli (1). Protein A fusions expressed in E. coli can thus be efficiently secreted to the periplasm of the bacteria, and in many cases also to the culture medium (50,53,55,85,88) from which they can be easily purified by IgG affinity chromatography (88,87). In the vectors designed for secretion, the expressed SpA-fusion products are in most examples transcribed from the SpA promoter, which is constitutive and efficiently recognized in E. coli (27,50).

Analogous expression vectors for production of secreted fusions also to the serum albumin-binding regions of SpG have been developed (14,50), taking advantage of the same SpA promoter and secretion signals. Fusion proteins produced in this way can be efficiently affinity purified using human serum albumin (HSA) columns (14).

There exists no general strategy applicable to all recombinant gene products to guarantee secretion. Instead, the inherent properties of the target protein largely dictate if a secretion route is possible to use. However, a large fraction of the proteins produced as SpA fusions are soluble and efficiently secreted to the periplasmic space and culture medium of E. coli (2,55,88). This may at least be partially caused by the high solubility of the ZZ domains, which most likely increases the overall solubility of the fusion proteins. There are a number of advantages connected with a secretion strategy. First, the gene product will be less exposed to cytoplasmic proteases, which might enable production of also labile proteins (88). Second, disulfide bond formation, which is occurring in the nonreducing environment outside the cytoplasm, could improve on the folding of certain gene products (88). Third, the recovery of the recombinant protein is simplified because a large de gree of the purification from host cell proteins has been achieved through the secretion (55,86). Hansson and coworkers (55) described the E. coli production of a fusion protein ZZ-M5, a malaria subunit vaccine candidate, using a secretion strategy. More than 65% of the recombinant gene product was found to be secreted to the culture medium, from which it could be recovered in a single step by expanded-bed anion exchange adsorption. As the ZZ-M5 protein was to be used in a preclinical malaria vaccine trial in Aotus monkeys (89), a polishing step was included in which the IgG-binding capacity of the fusion protein was employed. After affinity chromatography on IgG-Sephar-ose, contaminating DNA and endotoxin levels were well below the demands set by regulatory authorities. The overall yield of the process, performed in pilot scale, exceeded 90%, resulting in 550 mg product per liter of culture (55).

As mentioned earlier, there is no guarantee that a certain protein can be secreted even if an expression system with secretion signals is chosen for the production. Proteins with a strong tendency to precipitate intracellularly and proteins containing hydrophobic transmembrane regions may be extremely difficult to secrete (27,80). Intra-cellular expression of such proteins could be a more attractive alternative because of recent advances in in vitro renaturation of recombinant proteins from intracellular precipitates, that is, inclusion bodies (90). The mechanism for inclusion body formation is not fully elucidated, but expression driven from exceptionally strong promoters such as the T7 promoter (91), leading to very high expression levels, seems to increase the tendency for intracellular aggregation of the produced recombinant protein (90). Production by the inclusion body strategy has the main advantages that the recombinant product normally is protected from proteolysis and that it can be produced in large quantities. Levels up to 50% of total cell protein content have been reported (90).

Vectors for intracellular production of SpA fusions have thus been constructed in which the transcription is under the control of inducible promoters, such as lacUV5 (11), 1PR (11), trp (92,93), trc (94), or T7 (13), enabling a more controlled expression. Also, for intracellular production of BB and ABP fusion proteins, inducible vector systems have been used, employing trc (94), trp (51,93), or T7 (13) promoters, respectively. The most robust and in addition the most tightly controlled of these systems seems to be the T7 RNA polymerase-regulated promoter system (91). Using this system, Larsson and coworkers (13) recently described the expression of five different mouse cDNAs as fusions to ZZ and ABP, respectively. Expression levels ranged from 4 to 500 mg gene product per liter of culture.

Intracellularly produced and still soluble fusion proteins can be released by sonication or high-pressure ho-mogenization of cells before purification by IgG or HSA affinity. However, precipitated gene products need to be solubilized before the affinity purification procedure. It was recently demonstrated that affinity purification indeed can be useful also for the recovery of proteins with a strong tendency to aggregate during renaturation from inclusion bodies following standard protocols (93). Murby and coworkers (93) devised a recovery scheme for the production of such ZZ and BB fusions, applied on various frag ments of the fusion glycoprotein (F) from the human respiratory syncytial virus (RSV). Earlier attempts to produce these labile and precipitation-prone polypeptides in E. coli had failed, but several different F fusion proteins could by this novel strategy be produced and recovered as full-length products with substantial yields (20-50 mg/L). Because it was demonstrated that the IgG and HSA affinity ligands both were resistant to 0.5 M guanidine hydrochlo-ride, efficient recovery from inclusion bodies of the ZZ-F and BB-F fusions could be achieved by affinity chromatog-raphy in the presence of the chaotropic agent throughout the purification process (93). The described strategy has so far been successfully evaluated for a number of proteins of low solubility and should be of interest for efficient recovery of other heterologous proteins that form inclusion bodies when expressed in a bacterial host.

It is, of course, almost impossible to give general information concerning the production levels for the described expression systems because production levels are strongly dependent on inherent properties of the target gene products. It is however evident that both the secretion strategy and intracellular protein production in E. coli using the SpA and SpG expression systems can give high expression levels. For the secreted production, expression levels of 0.5 to 0.8 g product per liter of culture have been reported (55,95), and for intracellular production levels up to 3 g gene product per liter of culture have been obtained (96). The SpA and SpG expression systems are thus for many gene products a good choice because high expression levels can be obtained and efficient affinity purification of produced fusion proteins can be easily achieved.

Mild Elution of Protein A and Protein G Fusions by Competition

One possible drawback associated with the SpA and SpG affinity purification systems has been the need for elution by low pH from the affinity column. The elution of fusion proteins at pH 3, used routinely, has for some products been destructive, yielding biologically inactive products. By a recently presented concept, the low-pH elution can be circumvented by competitive elution based on an engineered competitor protein that can be efficiently removed from the eluate mix after elution. The principle for the competitive elution strategy was described by Nilsson and coworkers (47) (Fig. 2). The target protein in the presented examples was produced as a fusion to a monovalent Z-domain of SpA. A sample (e.g., crude cell lysate) containing the recombinant fusion Z-target is passed through an IgG-Sepharose column. The recombinant fusion protein is thus captured, enabling extensive washing to remove contaminants. A stoichiometric excess of an engineered bifunc-tional competitor fusion protein ZZ-BB (Fig. 2) was added in which BB constitutes a second affinity tail capable of selective binding (association constant; KA ^ 1.4 X 109 M_1) to HSA. The competitor ZZ-BB, having an affinity to human IgG more than 10-fold higher than that to the monovalent Z, is used for competitive elution of the Z-fusion at neutral pH. Specific removal of ZZ-BB from the effluent mix is accomplished by a passage through a second affinity column (HSA) to which the ZZ-BB fusion is bound.

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