PROTEIN EXPRESSION & ANALYSIS

Advances in industry and medicine have led to the engineering of complex “designer” proteins, such as antibodies in targeted therapeutics and enzymes in process development. The ability to easily generate an almost infinite number of variants at the DNA level has increased the demand for improved protein expression methodologies to fully capture what can be produced genetically. Often, the protein of interest is eukaryotic in origin and may require posttranslational modifications specific to its native host or may be toxic to the host cells expressing them. Cell-free protein expression systems have allowed us to step beyond the limits of traditional in vivo expression methodologies by decoupling protein expression from host cell viability (1,2,3). Furthermore, the ability to produce complex proteins using cell-free transcription/translation systems uniquely enables high-throughput directed evolution and protein engineering efforts (4,5). Several cell-free protein expression systems have been developed in the last decade with recent advances focusing on special folding or assembly environments (6,7,8). Equally as important is the capability to transition from the in vitro system to largerscale in vivo expression, while maintaining activity of the target protein (9,10).


Introduction
Advances in industry and medicine have led to the engineering of complex "designer" proteins, such as antibodies in targeted therapeutics and enzymes in process development. The ability to easily generate an almost infinite number of variants at the DNA level has increased the demand for improved protein expression methodologies to fully capture what can be produced genetically. Often, the protein of interest is eukaryotic in origin and may require posttranslational modifications specific to its native host or may be toxic to the host cells expressing them. Cell-free protein expression systems have allowed us to step beyond the limits of traditional in vivo expression methodologies by decoupling protein expression from host cell viability (1,2,3). Furthermore, the ability to produce complex proteins using cell-free transcription/translation systems uniquely enables high-throughput directed evolution and protein engineering efforts (4,5). Several cell-free protein expression systems have been developed in the last decade with recent advances focusing on special folding or assembly environments (6,7,8). Equally as important is the capability to transition from the in vitro system to largerscale in vivo expression, while maintaining activity of the target protein (9,10).
The PURExpress ® In Vitro Protein Synthesis Kit, supplemental PURExpress Disulfide Bond Enhancer, and SHuffle ® Competent E. coli from New England Biolabs provide a seamless system for in vitro to in vivo protein expression. PURExpress is a novel cell-free transcription/translation system reconstituted from the purified components necessary for E. coli translation. The nuclease-free and protease-free nature of the PURExpress platform preserves the integrity of DNA and RNA templates and results in proteins that are free of modification and degradation. The PURExpress ® Disulfide Bond Enhancer (PDBE) is a blend of proteins and buffer components designed to correctly fold target proteins with multiple disulfide bonds produced in PURExpress reactions. These enhancements can increase the yield of soluble and functionally active complex proteins containing disulfide bonds. Target proteins expressed in the PDBE/PURExpress environment can then be transitioned to in vivo expression using SHuffle E. coli strains. These engineered strains are capable of expressing proteins with increasing disulfide bond complexity in the cytoplasm. SHuffle strains express a disulfide bond isomerase that isomerizes mis-oxidized substrates into their correctly folded state, greatly enhancing the fidelity of disulfide bond formation. When used in conjunction, these three products increase the likelihood of generating fully active, complex proteins with multiple disulfide bonds.
Cellulases are glycoside hydrolases that catalyze the cleavage of β-1,4-D-glycosidic linkages in cellulose, a linear polymer of glucose units. Cellulases are often multidomain proteins consisting of a catalytic core, a flexible linker and a carbohydrate-binding module. Bacterial and eukaryotic cellulase mixtures perform key functions in the conversion of lignocellulosic biomass into fermentable sugars for renewable chemical and biofuel production, textile processing, and detergent formulations. EG3 from the fungi Humicola insolens, an endoglucanase that creates internal glucan chain scissions, is a multi-domain 42 kDa protein containing a carbohydrate-binding module (CBM1) having two nonconsecutive disulfide bonds connected by a flexible linker to a catalytic core (GH5) with one disulfide bond (based on the predicted crystal structure; See Figures 1 and 2). This enzyme is used as a detergent cellulase for color brightening, softening and soil removal. The capabilities of the PDBE/ PURExpress system with downstream expression in SHuffle E. coli strains are well-matched to the expression of this complex fungal protein in a flexible E. coli-based system. Assemble reactions on ice in nuclease-free, 0.5 ml microcentrifuge tubes as indicated*:

in vivo Expression:
To produce larger quantities of purified enzyme, clone the E3 gene into a suitible expression plasmid and transform into SHuffle using the protocol found on the NEB website (http://www.neb.com/ nebecomm/products/protocol390.asp). Plate onto antibiotic selection plates and incubate for 24 hours at 37°C. Resuspend a single colony in 3 ml LB containing antibiotic and grow the starter culture overnight at 37°C. Inoculate 50 ml MagicMedia ™ (Life Technologies) with 50 µl of the starter culture and grow in 250 ml baffled flasks at 37°C until reaching 1 OD, upon which transfer to growth at 25°C for a total of 24 hours.
Harvest cells by centrifugation at 3,000 rpm for 30 minutes at 4°C. Resuspend cell pellets in BugBuster Plus solution containing protease inhibitors and lysozyme [see note 1, page 3] at 4 ml/g wet cell mass. Lyse for 30 minutes according to the manufacturers protocol. Affinity purify EG3 from the lysate (e.g. immobilized metal affinity purification), quantitate by using absorbance at 280 nm, and assess for purity by SDS-PAGE. * Refer to current PURExpress (http://www.neb.com/nebecomm/products/productE6800.asp) and PDBE (http://www.neb.com/nebecomm/products/productE6820.asp) manuals for latest protocols, as recommended volumes and incubation times have been updated.
** Typical starting amounts are 1 µl for both PDBE 1 and 2. Titration of reagent may be necessary.  These tools can be utilized in a general workflow where the high throughput screening and selection of variants of a complex protein of interest can be done in a cell-free environment ( Figure 5). The successful variant can then be expressed in vivo by using a specialized E. coli strain that also facilitates the required protein folding needs. In this case, the formation of complex disulfide bonds with the correct pairing was crucial to successful expression of active EG3 in both in vitro and in vivo expression environments.