Scale Down of Intein Processes

As mentioned above, inteins are expected to have wide use in proteomics research for recovery, isolation and analysis using miniaturized channels on a microfluidics platform and for orienting and tagging proteins in microarrays and on chips. Scaling down intein technology will have application in meeting the requirements of fast, low-volume, and parallel processing for proteomics analysis. Miniaturization is a key component with any high-throughput technology and requires substantial engineering input. Drug discovery, sensors development, and assay integration are examples of potential applications of this technology to biomedicine, biodefense, and biotechnology. After summarizing why inteins could be useful in microfluidics, we describe the use of inteins in recovering and detecting proteins in miniaturized fluidics channels. Then, in a second application of inteins, we illustrate the use of intein-mediat-ed biotinylation of proteins for protein microarray analysis and screening.

3.1 Microfluidics

The main goal of microfluidics devices for scale-down bioprocessing is to transfer, recover and analyze very small quantities of fluids containing a desirable protein on a miniaturized platform, i.e., chip or disk, such that the final product is of high yield and purity. In some cases it may be necessary to crystallize the final product. As Fish and Lilly (1984) have shown with large-scale bioprocessing for the production of pharmaceuticals, the fewer the number of isolation and recovery steps or unit processes the better, i.e. less product is lost overall. Hence, adsorptive processes and specifically bio-affinity capture techniques are particularly attractive since they exhibit high selectivity and can be easily miniaturized (small beads with high ligand densities).

For small-scale massively parallel applications on a fluidics platform, it is imperative that the purification process be as simple as possible with a minimum number of recovery steps and minimum addition of cofactors or other chemicals. Tripartite fusion proteins, with inteins as controllable linkers, binding domains for selective affinity adsorption and the protein of interest, are attractive for microfluidics bioprocessing for the following reasons:

1. Cleavage or splicing is non-protein-specific, i.e., the same controllable intein linker can be used with different product proteins. This broadens the applicability of the process to any binding domain and product of interest.

2. Cleavage or splicing occurs without additives or cofactors (besides hydrogen ions for temperature/pH-controllable inteins) and does not require exogenous proteases. Also, external triggers such as temperature can be universally used to simultaneously cleave desired proteins from their fusions.

3. Binding of the tripartite fusion to the solid substrate (bead or synthetic microporous membrane) is specific for a chosen binding domain, yet generalizable for all product proteins, i.e., the same ligand-adsorbent can be used for different proteins.

Miao et al. (2005) have reported on a single-step fusion-based affinity purification of proteins from E. coli lysates with pH-controllable linkers and a chitin-binding domain (CBD) in a fluidic device. The linkers were previously derived from self-splicing inteins (Wood et al. 1999). Two different linkers were generated to solve two distinct separation problems: one (called the CM or cleaving mutant mini-intein) for rapid single-step affinity purification of a wide range of proteins (Wood et al. 2000), and the other (called the SM or splicing mutant mini-intein) specifically for the purification of cytotoxic proteins (Wu et al. 2002). The two problems addressed by scale-down of the intein cleavage and splicing in a fluidics channel are protein purification by intein-mediated cleavage and purification of a cytotoxic protein by insertional inactivation and protein splicing (Fig. 5A,B). A rotating compact disk (CD) format was chosen due to its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design of the fluidic device is shown in Fig. 6. The fluidics platform was based on standard photolithography and wet chemical etching techniques of a silicon substrate to produce a fluidics channel with two reservoirs at each end and containing a barrier that retained the beads but allowed the fluid to pass. The silicon channel was covered and sealed with a film of silicone rubber [poly(dimethyl siloxane), PDMS] and angled holes drilled through the PDMS above the reservoirs. Several channels and covers were then adhered to a blank CD, placed in a rotating device, filled with affinity beads and E.coli lysate in the inner reservoir, and spun to drive (pump) the fluid through the affinity bed to the outer reservoir. An elution buffer was then passed through the bed and the recovered proteins analyzed. Scale-down factors of ~200-fold were achieved by separating in a -25-^1 bed volume. This work is widely applicable to small-scale massively parallel proteomic separations.

After expressing the tripartite fusion protein (step 1 in Fig. 5A), the clarified cell lysate is loaded onto a chitin column and allowed to selectively bind to the column (step 2). Then the pH is shifted so that the intein (CM) selectively cleaves at its C-terminus (step 3), allowing the purified product protein to be released into the eluate. This fluidic separation has been applied to the a-subunit of RNA polymerase and to the DNA-binding domain of the intron endonuclease I-Tevl.

In the second example of single-step affinity purification and recovery on a microfluidics platform from an E. coli lysate, a toxic protein was recovered directly from a fluidics column by selective controlled splicing of the linker domain from a tripartite fusion, allowing post-assembly of the desired toxic protein (Wu et al. 2002). In this case, the toxic protein was inactivated by insertion of SM, the controllably splicing mini-intein. Affinity purification was then achieved via the CBD which was itself inserted into the SM (Wu et al. 2002; Fig. 5B). Purification steps 2 and 3 are essentially the same as those used for the intein cleavage process. Again, microfluidic eluate was compared with material purified in a column, and found to be of equivalent purity and specificity. This fluidic separation was applied to the toxic intron endonuclease I-Tevl. The yield of protein from the microfluidics platform was approximately equivalent to that from the laboratory-scale preparation (Wu et al. 2002). Also, the endonuclease activity of I-Tevl from the fluidics purification exhibited approximately the same activity as that using a laboratory column. These experiments demonstrated not only that this cytotoxic protein can be purified at small scale, but also that the purified protein is highly active.

Fig. 5. Intein-mediated purification systems and oriented immobilization of proteins through intein-mediated biotinylation on a fluidics platform, a Strategy for purification of a target protein (P) using controllable CM intein (J) cleavage with a chitin-binding domain (CBD) in a tripartite fusion protein. Step 1 Expression of the tripartite fusion precursor protein in vivo. Step 2 After cell lysis and clarification in chitin column buffer (pH 8.5), the cell lysate is loaded onto a chitin column. Step 3 After washing the column, the pH is shifted from 8.5 to 6.0 to trigger C-terminal cleavage and the cleaved products are eluted after 24 h at 4°C. b Strategy for intein-mediated purification of a cytotoxic protein (N-C) using

Fig. 5. Intein-mediated purification systems and oriented immobilization of proteins through intein-mediated biotinylation on a fluidics platform, a Strategy for purification of a target protein (P) using controllable CM intein (J) cleavage with a chitin-binding domain (CBD) in a tripartite fusion protein. Step 1 Expression of the tripartite fusion precursor protein in vivo. Step 2 After cell lysis and clarification in chitin column buffer (pH 8.5), the cell lysate is loaded onto a chitin column. Step 3 After washing the column, the pH is shifted from 8.5 to 6.0 to trigger C-terminal cleavage and the cleaved products are eluted after 24 h at 4°C. b Strategy for intein-mediated purification of a cytotoxic protein (N-C) using

3.2 Protein Micro-arrays

During this post-genomic era, attention is being focused on the proteome in order to understand the role of proteins in vivo and to develop drug targets, biomarkers and treatment tools for various diseases. Screening methods that rely on bead-based and biochip technologies are offered as possible solutions. The efficacy (sensitivity, reproducibility and reliability) of the latter processes depends on the efficient capture of proteins on solid substrates. Strategies for orienting proteins or peptides to substrates are often very tedious, requiring many steps, and binding to the surface (ligand) is often not very tight or selective (such as with His-tag to Ni-NTA). Inteins can also be used to modify proteins so that they can attach to solid substrates in a desirable orientation. Yao and his group have reported on the use of intein-mediated site-specific biotinylation of proteins (maltose-binding protein, enhanced green fluorescent protein, and glutathione S-transferase) in vitro and in vivo and subsequent immobilization onto avidin-functionalized glass slides (Lesaicherre et al. 2002; Lue et al. 2004). Their process is summarized in Fig. 5C. After expression and loading of the tripartite fusion protein onto a chitin column (steps 1 and 2), cleavage at the N-terminus of the intein through addition of cysteine-biotin (step 3) releases the biotin-conjugated protein for immobilization onto an avidin-coated substrate (step 4). Based on SDS-PAGE, the efficiency of the biotinylation was 90-95%. The strategy generated "a protein array on which the proteins were oriented optimally and were able to retain their native activity suitable for subsequent biological screening" (Lesaicherre et al. 2002).

controllable SM intein splicing with CBD in a tripartite fusion protein. Step 1 The intein-disrupted non-toxic precursor protein is expressed in vivo after induction with IPTG. Step 2 After cell lysis and clarification in chitin column buffer (pH 8.5), the cell lysate is loaded onto a chitin column. Step 3 After washing the column, the pH is shifted from 8.5 to 7.5 to trigger splicing. The products are eluted after 24hat 4 °C. They include full-length I-Te-vl and the N- and C-terminal cleavage products (after Miao et al. 2004). c Strategy for purification, reaction (biotinylation) and oriented immobilization of a target protein (P) using controllable N-terminal intein (J) cleavage with a chitin-binding domain (CBD) in a tripartite fusion protein. Step 1 Expression of the tripartite fusion precursor protein in vivo. Step 2 After cell lysis and clarification in chitin column buffer, the cell lysate is loaded onto a chitin column. Step 3 After washing the column, cysteine-biotin (Cys-biotin) is added to the column buffer to trigger N-terminal cleavage and to covalently attach biotin (B) through the cysteine reaction to the protein. Step 4 After eluting the cleaved products from the column, they are added to an avidin-coated glass slide to generate a protein microar-ray. (After Lesaicherre et al. 2002)

Fig. 6. Fluidics channel on a rotating compact disk (CD) for intein-mediated protein purification. a Plan view of the scaled-down fluidic channel on a rotating CD with poly(dimethyl siloxane) (PDMS) covers and filled with affinity beads. The direction of fluid flow is from the loading (feed or inner) reservoir to the eluting (product or outer) reservoir (arrow) and is driven by centrifugal force. The reservoirs at both ends of the channel are drilled at an angle in the PDMS cover to reduce liquid overflow. The rotational speed is 250 rpm. b Optical micrograph of the fluidics channel with 50-100 |im diameter chitin-coated affinity beads. The depth of the column is 0.6 mm. Other dimensions are given in the figure

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