Endoproteases specific for cleavage of peptidyl bonds on the C-terminal side of lysine residues (e.g., Lys-C) are produced from a number of bacterial species, including Achromobacter lyticus (1), Pseudomonas aeruginosa (2), and Lysobacter enzymogenes (3). The Achromobacter protease 1 (API) protein has been substantially characterized (4,5,6) and shown to be a resilient enzyme that can specifically cleave after lysine residues under a wide range of buffer conditions, including high concentrations of denaturing agents such as urea and sodium dodecyl sulphate (SDS). Those properties make this protease particularly useful for peptide mapping (3) and in-gel digestion (7) applications.

PRODUCT FOCUS: PROTEINS



PROCESS FOCUS: DOWNSTREAM PROCESSING



WHO SHOULD READ: R&D, PROCESS DEVELOPMENT, ANALYTICAL, AND MANUFACTURING PERSONNEL



KEYWORDS: LYSOBACTER ENZYMOGENES, LYS-C, ANIMAL-FREE MEDIA, PLANT-BASED EXTRACTS, DISPOSABLE BAG FERMENTORS, PROTEASE PURIFICATION, SEQUENTIAL CHROMATOGRAPHY, ENZYME CHARACTERIZATION



LEVEL: ADVANCED

Starting with the Lysobacter enzymogenes American Type Culture Collection (ATCC) strain 27796, we developed an animal-free media composition by optimizing both the plant-based protein extract source and sugar type. Then we scaled up the Lysobacter culture to 40 L using a Cellexus fermentor with disposable bag technology. Various purification schemes for Lys-C have been published involving several chromatography steps and various precipitation techniques (4, 8, 9). All those purification schemes take multiple days and involve numerous concentration/dialysis steps as well as cold temperatures to give good protease recovery with little loss due to self digestion. Here, we combine the different attributes of four chromatography media to elicit rapid purification of the endoprotease Lys-C from culture supernatant. By sequentially loading the elution fractions from one column directly (or with minimal sample handling) onto the next column in the series, we reduced the purification time and therefore self digestion of the protease compared with other methods. Our process yielded pure Lys-C protease with specific activity towards lysine residues, a low endotoxin level, stable storage characteristics, and good tolerance to freeze–thaw cycles. We produced a final Lys-C preparation without any contact with animal products, and the process can be scaled to produce gram quantities of the enzyme.

A complete amino acid sequence of the isolated Lys-C protein was determined by cloning and sequencing the gene from Lysobacter strain ATCC 27796. We identified a new isoform of Lys-C that shares mixed identity with both the Lep A and Lep B genes already published for Lys-C proteases derived from Lysobacter (9, 11).

Materials and Methods

Culture Conditions: We grew Lysobacter enzymogenes subspecies enzymogenes (ATCC 27796) cultures in an animal-free medium containing 1% HiVeg plant extract from HiMedia Laboratories (www.himedialabs.com), 1% glucose, 0.01% of each mono- and dibasic potassium phosphate, and 0.02% magnesium sulphate. All other tested media substitutions are indicated in the results section, below. We used shake-flask cultures at 50-mL and 1-L volumes to generate a 1-L fermentor inoculum. The initial OD₆₀₀ measurement for each shake flask culture was 0.05 ± 0.01. Cultures grew at 30 °C with 225 rpm shaking, and final OD₆₀₀ measurements of 3.5–4.5 and 2.5–3.0 were achieved after ∼24 hours for the 50-mL and 1-L cultures respectively.

The fermentor medium's carbon source was supplemented to a level of 2% (w/v) glucose. Fermentation cultures were initiated at a starting OD₆₀₀ of 0.06–0.07 and reached a final OD₆₀₀ of 4.5–5.5 after ∼72 hours at 30 °C with 8-L/min aeration. Fermentation was carried out in a Cellmaker Lite 2 from Cellexus Inc. (www.cellexusbiosystems.com), using disposable CellexusBag technology for aeration and mixing. Our assembly followed the manufacturer's recommendation using filters for sterile gas exchange and media loading as well as a sterile sample/injection port assembly. All sterile connections to the fermentor bag were made in a laminar flow hood.

Enzyme Purification: From the 40-L fermentation culture, we clarified 36 L using batch centrifugation in a Beckman/Coulter Avanti J-20 XP centrifuge (www.beckman.com) with a JLA 8.1000 rotor at 15,900_g for 15 minutes. Then we collected the culture media and passed it in 11-L batches through a 0.45-µm filter from Pall Corporation (www.pall.com) onto a BPG-200 column packed with 2 L of DEAE-Sepharose media from GE Healthcare Bio-Sciences AB (www.gehealthcare.com) using a 620S peristaltic pump from Watson-Marlow Bredel (www.watson-marlow.com). The flow-through volume was collected and the active protease precipitated with 70% (w/v) ammonium sulphate for an hour at 4 °C with stirring.

Following centrifugation of each batch using the same centrifuge and rotor at 15,900_g for 30 minutes, the protein pellet was solubilized in 1 L of 1-M NaCl and 20-mM Tris at pH 8.0, then loaded onto an XK 50 column from GE Healthcare packed with 100 mL of PPA Hypercel resin from Pall equilibrated in the same buffer. We allowed a residence time of 10 minutes (with a 10-mL/min flow rate) to ensure complete binding before washing with three column volumes of buffer (1-M NaCl and 20-mM Tris at pH 8.0).

Lys-C was batch eluted with 8-M urea and 20-mM Tris at pH 8.0. A single peak containing protease activity (as determined by colorimetric assay) was pooled and loaded at 5 mL/min directly onto a reverse-phase Tricorn column from GE Healthcare packed with 9 mL of Resource 15 reverse-phase media, also from GE Healthcare. After the column was washed with two column volumes of 20-mM Tris at pH 8.0 and 10% acetonitrile, a 10–25% linear gradient of acetonitrile was initiated. Fractions containing protease activity were pooled, diluted twofold with 20-mM Tris at pH 8.0, and loaded directly onto a ϖ-AminoHexyl Sepharose column at 2 mL/min (an XK 16 column from GE Healthcare Bio-Sciences AB) packed with 8 mL of ϖ-AminoHexyl-sepharose media from Sigma-Aldrich Canada Ltd. (www.sigmaaldrich.com). The column was washed with 20-mM Tris at pH 8.0 and then eluted with a 0–700 mM linear gradient of NaCl. We observed a single peak that contained Lys-C protease activity and pooled the fractions containing the highest protease activity, then quantitated that activity by colorimetric assay. Samples were then aliquotted for storage at −20 °C.

Colorimetric Protease Assay: We monitored the Lys-C enzyme reaction rate against the substrate Ac-Lys-pNA (Bachem Catalog #L-1045, www.bachem.com) at 405 nm over a period of 30 minutes using a SpectraMax Plus 384 microplate reader from Molecular Devices (www.moleculardevices.com). Each 200-µL Lys-C reaction included 185 µL of 180-mM Tris-HCl at pH 9.0, 250-µM Ac-Lys-pNA, and 15 µL of test sample. We measured the reaction rate as a change in 405-nm absorbance over time (mOD/min) and extracted the maximum slope from the resulting curve.

Lys-C Characterization Assays: We determined Lys-C proteolytic activity at different pH values (4,5,6,7,8,9,10,11,12,13) using the colorimetric assay as above. The substrate at 250 µM in 200-mM Tris was mixed with 5 µL of Lys-C. (Various pH values were generated by mixing 200-mM Tris-HCl with 200-mM Tris-Base, and for the very high pH values 10-M NaOH was added.) We assessed protease activity at each pH value by noting the initial rate of the proteolytic reaction. We similarly determined the effects of other additives (urea, guanidine hydrochloride, NaCl, acetonitrile, and SDS) by preparing a series of buffers containing those additives and then adding a stock solution of substrate, then initiating a reaction by addition of the Lys-C. To determine the enzyme kinetics, we prepared various buffers containing different amounts of the colorimetric substrate. We estimated the Michealis constant (K_m) and V_max values from a Lineweaver-Burke plot. Repeating this analysis with increasing concentrations of different inhibitors helped us determine the inhibition constant (K_i) for competitive inhibitors.

Cloning and Gene Sequencing: We assembled the gene sequence encoding Lys-C from L. enzymogenes (ATCC 27796) from three independent but overlapping fragments amplified by polymerase chain reaction (PCR). For the first fragment, a forward primer (5′-GGCCAGTGCAACGTCGAC GTGGTCTGCC-3′) was designed based on an N-terminal amino-acid sequence (GVSGQCNVDVVCP) recovered from the purified protein. This was combined with a reverse primer (5′-GTGCGGTTGTGCAG GTTGTAGAC-3′) designed to a region conserved within the C-extension domain in both lepA (Genbank Accession AB045676) and lepB (Genbank Accession AB094439) genes from Lysobacter species IB-9374. That fragment provided the DNA sequence of the mature protease coding sequence and a portion of the C extension.

To isolate the N-terminal prepro sequence, we designed a reverse primer within the recovered sequence of the first fragment (5′-GATAGTTCCAATACACCACGATG-3′) and paired it with a forward primer (5′-CTTCAAACGCACGCTGTAGGGGAAG-3′) designed from the published lepA promoter region that spans the 35–10 region. The third C-terminal fragment was isolated by chromosome walking using a single specific primer to the C-extension region (5′-CGATCTACCACACCTACAAGAG-3′).

Each PCR used 50 ng of sonicated Lysobacter genomic DNA as a template and included 1× Q-solution from Qiagen (www.qiagen.com) in the reaction to facilitate amplification from the high-percentage guanine–cytosine DNA of Lysobacter. We cloned the PCR products into a pGEM-T vector for sequencing. DNA sequencing of the lys-c gene on both strands using the dideoxy chain termination method (12) performed at the University of Calgary Core DNA Sequencing Laboratory (www.ucalgary.ca/dnalab). The resulting DNA sequence and its deduced amino-acid sequence were analyzed using Vector NTI software (www.invitrogen.com).