The PlyCP41 gene (GenBank KX884995) was synthesized as an E. coli codon optimized construct and cloned into pET21a (Novagen®, Millipore Sigma, Billerica, MA) by GenScript (Piscataway, NJ) . PlyCP41 contains a C-terminal 6xHis-tag to facilitate purification using an Ni-NTA resin. pET21a: PlyCP41 was transformed and maintained in E. coli TOP10 cells (Life Technologies, Carlsbad, CA). The plasmid was transformed into E. coli strain BL21 (DE3) (Stratagene, La Jolla, CA) for protein production. As a control for expression in bacteria, plasmid pET21a: Lysin D (unpublished), which encodes another bacteriophage endolysin of similar size, was also transformed in BL21 cells for protein production only in E. coli. A plant codon-optimized CP41 gene containing a C-terminal 6xHis-tag (PlyCP41pc) with a CAI index of 0.92 was synthesized by Genscript USA (Piscataway, NJ) and was cloned in the pJET1.2 vector  (Additional file 1: Figure S1).
For expression in plants, the PlyCP41 coding region was amplified from the pET21a vector using primer pair BKEYS08 (5′- CCGGATCCAACAATGCTGAAGGGTATCGACGTTAGC-3′) and BKEYS09 (5′- CCAAGCTTTCAGTGGTGGTGGTGGTGGTGCTCGAG-3′) and AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) and the amplicon was cloned in the pCR4 vector (Life Technologies) for sequence analysis. The gene was then isolated by restriction digestion using BamHI and HindIII and inserted into a similarly digested intermediate pSKAS vector that is based on pBluescript SK+ and containing nt 4945 to nt 6541 of the pP2C2S PVX-based vector  and an expanded multiple cloning site . The intermediate pSKAS vector allows engineering of insertions into a smaller plasmid vector from which the insertion can be transferred into the full-length virus-based vector. The resulting pSKAS: CP41 plasmid was digested with ApaI and SpeI and the insert was isolated and cloned into the similarly digested pGDPVXMCS plasmid (containing the full-length, PVX genome ), creating pGDPVXMCS: CP41p. The E. coli codon-optimized gene was maintained in this construct.
The plant codon-optimized PlyCP41pc gene was amplified from pJET: CP41p using oligonucleotide primers BKEYS17 (5′- CCCATGGAACAATGCTTAAGGGAATTGATGTTTCTGAAC-3′) and BKEYS18 (5- CCGAATTCCTAATGATGATGATGATGATGAAGTTTC 3′). The resulting amplicon was cloned into pCR4 vector. The PlyCP41pc gene was isolated from pCR4:CP41p by digestion with NcoI and EcoRI and cloned into the NcoI/EcoRI sites of the pSKAS vector, creating pSKAS: PlyCP41pc. Digestion of this plasmid with ApaI/SpeI released a fragment that was ligated into ApaI/SpeI digested pGDPVXMCS, creating pGDPVXMCS:PlyCP41pc. For all cloning, PCR products and gene fragments were gel purified from 1% agarose/TBE gels using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany), vectors and inserts were ligated using T4 DNA ligase (New England Biolabs), and transformed into competent Top 10 E. coli cells (Life Technologies). The plasmid constructs were maintained the E. coli TOP 10 cells using appropriate antibiotics and plasmid DNAs were purified using the QIAprep Miniprep kit (Qiagen GmbH). All plasmids were sequenced for verification (Genscript USA).
Bacterial protein overexpression and purification using the BugBuster reagent
The pET21A:PlyCP41 construct was transformed into E. coli BL21 (DE3) cells for protein induction. Briefly, 5 mL of LB broth was inoculated with a loop of bacterial cells harboring the construct. After overnight incubation in a shaking incubator at 37 °C, 100 μl of cells was inoculated into 2 mL of LB and the culture was grown for 2 h. An aliquot of the culture was removed as a non-induced control. For protein induction, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was then added to the cell cultures at a final concentration of 2 mM and the cultures were incubated with shaking at 37 °C for a further 2 h, during which time aliquots were removed at after 1 and 2 h for analysis by SDS-PAGE. For large scale protein purification of induced proteins, 500 μl of an overnight culture was added to 50 mL of LB in a 125 ml Erlenmeyer flask. Bacterial pellets were recovered from 50 mL of IPTG-induced bacterial cultures grown at 37 °C by centrifugation at 4000 x g for 20 min in a Jouan CR422 centrifuge (Saint-Herblain, France) as previously described . The BugBuster Master Mix Protein Extraction Reagent (Novagen, Madison, WI) and protease inhibitor cocktail for plant cells) (Sigma Chemical Co.) (1 μL cocktail per 100 μl of BugBuster Reagent) was added to the bacterial pellet to prepare the bacterial lysates and extract total proteins. The extraction was carried out per manufacturer’s instructions to obtain total, soluble, and inclusion body fractions. For determination of protein concentrations, the Bradford assay using the Quick Start™ Bradford 1xDye Reagent and Quick Start™Bovine Serum Albumin (BSA) Standard Set (Bio-Rad Laboratories, Hercules, CA) were used per manufacturer’s instructions.
Agroinfiltration of N. benthamiana leaves
Agrobacterium tumefaciens strain EHA105 was transformed with the pGDPVXMCS, pGDPVXMCS: PlyCP41p, and pGDPVXMCS:PlyCP41pc plasmids and the bacteria were plated on Luria Broth-glucose (LBg) agar containing rifampicin and kanamycin at 50 μg/mL each. Colonies which appeared after incubation of the plates at 28 °C were inoculated into 5 mL of liquid LBg broth and grown overnight at 28 °C and 250 rpm in a shaking incubator. The cultures were centrifuged for 10 min at 4000 x g at 25 °C in a Jouan CR422 centrifuge. The bacterial pellets were gently resuspended in 2 mL of infiltration medium (10 mM MES, 10 mM MgCl2, pH 5.7) and 4 μL of 1 M acetosyringone (Sigma Chemical Co.) was added. After incubation at ambient room temperature for 4 h, the cultures were individually mixed with a culture of similarly prepared EHA105 containing the plasmid pGDp19 (encoding a plant viral-encoded suppressor protein ) at a ratio of 1:10 (pGDp19: pGDPVXMCS construct). Three to four young leaves N. benthamiana plants at the 5–6 leaf stage were infiltrated on the abaxial side of the leaf using a needleless syringe. Plants were grown in the laboratory at 27 °C and were observed for symptom production and monitored for virus infection using PVX AgriStrips following manufacturer’s instructions (Eurofins BioDiagnostics, Inc., Longmont, CO).
CP41 extraction from N. benthamiana plants
Four leaf discs (~ 20 mg tissue) were collected and placed into an eppendorf tube to test for PlyCP41p and PlyCP41pc protein production. The leaf samples were ground in 100 μl of the CellLytic™ P Plant Cell Lysis/Extraction Reagent (Sigma Chemical Co., Saint Louis, MO) containing 1 μl of plant protease inhibitor cocktail (Sigma Chemical Co.) using a blue pestle. Cell debris was removed by centrifugation at 4 °C, and the supernatant was combined with an equal volume of Laemmli buffer (BioRad Laboratories, Hercules, CA). After boiling for 10 mins, an aliquot of the sample was applied to a 10–20% Tris-glycine gel as described below.
Protein gel electrophoresis and Western blot analysis
Proteins were resolved by SDS-PAGE analysis on a Novex 10–20% Tris-glycine gradient mini gels (Life Technologies) under denaturing conditions using manufacturer’s instructions. The proteins were visualized by staining with SimplyBlue Safe Stain (Life Technologies). Alternatively, the proteins were transferred to a 0.45 μM nitrocellulose membrane (Life Technologies). The membranes were subsequently incubated with a 1:1000 dilution of Anti-His HRP Conjugate solution (Penta His HRP Conjugate Kit (Qiagen) following manufacturer’s instructions followed by development using the TMP Membrane Peroxidase Substrate System (Kirkegaard and Perry, Gaithersburg, MD) to visualize the proteins.
Protein purification using nickel resin (IMAC) under native conditions
To purify bacterial and plant expressed His-tagged proteins under native conditions, we used the Ni-NTA His-Bind Resins Kit and the Ni-NTA Buffer kit (Novagen) following manufacturer’s instructions to purify PlyCP41 from the soluble fraction obtained previously from E. coli using the Bug Buster reagent (above). The plant PlyCP41p protein was also purified from plant sap using the Ni-NTA His-Bind Resin under native conditions. Plant tissue was ground in a chilled mortar and pestle using Binding Buffer (BB; 50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 10 mM imidazole) from the His-Bind Resins Kit, to which the plant protease inhibitor cocktail (Sigma Chemical Co.) was added in a ratio of 1 μl cocktail to 100 μl BB. The Bradford assay described above was used to determine protein concentrations in the fractions.
Testing the lytic activity of expressed proteins against C. perfringens
The plate lysis (spot) assay was performed essentially as described previously . C. perfringens strain Cp39 cultures were propagated to mid-log phase (OD600 = 0.4–0.6) in 50 mL BHIB, where upon the cells were centrifuged at 5000 g for 30 min. The cell pellet was washed with 50 mL lysin buffer (50 mM NH4OAc, 10 mM CaCl2, 1 mM DTT, pH 6.2) and pelleted again. The cells were suspended in 1.0 mL lysin buffer. Ten milliliters of 50 °C semisolid BYC ss agar (37 g/L brain heart infusion powder, 5 g/L yeast extract, 0.5 g/L cysteine, 7 g/L Bacto agar) was added to the cells and then the cells were poured into a sterile 6 × 6 grid square petri dish. The plates sat 20 min at room temperature to solidify the agar. Ten μL of the purified endolysin or plant sap was then spotted onto the plate and allowed to air dry 20 min. The purified lysins were in diluted in “10:90” buffer (50 mM NaH2PO4, pH 7, 30 mM NaCl, 2 mM imidazole, 3% glycerol). Plant sap was prepared from uninfected tissue, leaf tissue from plants infiltrated with the empty pGDPVXMCS, PVX-based vector, and plants infiltrated with the pGDPVXMCS: PlyCP41p plasmid by grinding 4 leaf discs (~ 20 mg) with a pestle in 100 μL 1 x PBS buffer (Bio-Rad, Hercules, CA) in an Eppendorf tube. After one round of centrifugation at 16,000 x g for 5 min, the supernatant was removed from the pellet containing cellular debris and 10 μL of plant sap was applied to the plate as described. A positive lytic reaction was determined by visible clearing of the turbidity of the bacterial cells. The plate was observed for development of visible clearing and then incubated overnight in an anaerobic chamber at 37 °C.