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Application Note

Simplifying the Bacterial Recombinant Protein Workflow while Maximizing Recovery
A Complete Solution from Expression to Detection
Introduction
Protein research encompasses many techniques, including structural analysis, enzyme kinetics and mechanism of action studies. For most of these techniques, it is crucial to produce and purify sufficient quantities of functionally active, highly pure, recombinant protein(s). Using the same batch of purified, recombinant protein across all structural and functional studies can help eliminate equivocal results generated by lot-to-lot variability in protein activity and sample composition. Strategies for recombinant protein expression involve transforming bacterial cells with a DNA vector encoding the protein of interest and then culturing the cells such that they transcribe and translate the desired protein. Typically, cells are then lysed to extract the expressed protein, which can then often be purified by exploiting its selective affinity for a particular immobilized ligand. Optimizing the performance parameters at each stage of protein production (expression, extraction, purification, detection) is paramount to ensuring reproducibly high protein yields, but the optimization process can be very time-consuming. Therefore, the commercial availability of complete sets of tools and reagents specifically designed to perform optimally at each step of protein production can greatly accelerate protein research.
Cell Culture and Expression Separation and Preparation Protein Detection

Protein Extraction BL21 (DE3) Cells Overnight Express™ Media BugBuster® Protein Extraction Reagent Benzonase® Nuclease rLysozyme™ Solution Protease Inhibitors

Affinity Purification PureProteome™ Magnetic Beads Direct Detect™ Spectrometer

Figure 1. Generic Protein Sample Preparation Pipeline. Optimized reagents and tools were used in this study to improve all the steps of recombinant protein production, including initial cell culture and expression, through separation and preparation, to protein detection.

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

The key parameters affecting recombinant protein expression during bacterial cell culture are cell density and induced protein load per cell. During the protein extraction phase, the key requirements are efficient disruption of bacterial cell walls, effective solubilization of proteins, and maintaining protein integrity and activity. It is vital to gently and quickly extract proteins to minimize exposure to degradative conditions. After cell lysis, purification of the recombinant protein is most efficient if the method used is compatible with the same buffering conditions used for extraction. Finally, researchers need simple detection platforms to quantify yield and assess purity of recombinant protein samples. A number of new reagents have been developed that optimize each stage of recombinant protein production. The Overnight Express™ auto-induction media eliminate the need to monitor bacterial culture density and do not require IPTG (isopropyl-b-d-1-thiogalacto-pyranoside) induction, thereby reducing labor and time, while often increasing expression levels. For gentle, but efficient, lysis, BugBuster® Extraction Reagent, a combination of detergents and other ingredients, enables non-mechanical extraction of soluble proteins from bacterial cells. Two enzymatic agents, Benzonase® nuclease and rLysozyme™ solution, have been shown to vastly improve sample recovery. For protein purification, the PureProteome™ magnetic bead systems offer low non-specific binding with minimal sample loss. Lastly, the Direct Detect™ IR-based quantitation system enables assay-free measurement of proteins without the limitations of colorimetric assays or ultraviolet/visible (UV/Vis)-based quantitation. The results presented in this application note aim to assess the performance of each of these reagents whether used alone or in combination.

Methods
Bacterial Culture
Transformation 6XHIS-CRP (c-Reactive Protein) plasmid DNA was isolated using the Montage® Plasmid Miniprep96 Kit (EMD Millipore Cat. No. LSKP09624). 10 ng of DNA was added to 20 µL competent cells (BL21[DE3], EMD Millipore Cat. No. 69450-4) in chilled 1.5 mL tubes. The tubes were heated at 42 °C for 30 s and placed on ice. 80 µL of SOC medium was added and mixed gently. 10 µL of cells were plated onto Luria Broth (LB) agar plates (supplemented with 100 µg/mL ampicillin [AMP]) and incubated at 37 °C overnight (O/N). Protein Expression 50 mL sterile medium (1x LB + 100 µg/mL AMP (LB-AMP)) was inoculated with one 6XHIS-CRP colony. The culture flask was incubated at 37 °C with 300 rpm shaking until A600 reached 0.5 to 1.0. Two methods of induction were used: (1) 12.5 mL of log-phase starter culture was added to 500 mL LB-AMP and grown as above until A600 reached 0.5 to 0.7 (approx. 2.5 h). Expression was induced by growing cultures for an additional 4 h in presence of 1 mM IPTG and (2) 12.5 mL starter culture was added to 500 mL of Overnight Express™ instant LB medium (EMD Millipore Cat. No. 71757-4) + 100 µg/mL AMP. Cultures were incubated at 37 °C with 300 rpm shaking for 16 h. Cell Harvest Cultures were dispensed (50 mL aliquots) into preweighed 50 mL tubes and pelleted by centrifugation at 5000 rpm for 20 minutes. The supernatant was discarded and wet cell pellet mass determined using a balance. Pellets were frozen at -20 °C until lysis.

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Cell Lysis
Cell lysis was performed using both chemical (buffer only) and mechanical (with sonication) methods using various buffer conditions. Lysis buffers included: (1) BugBuster® protein extraction reagent (BB, EMD Millipore Cat. No. 70584-3); (2) Homebrew (HB): 20 mM Tris-Cl, pH 8, 137 mM NaCl, 10% glycerol, 1% NP40, pH 8. HB lysis buffer was filtered using a 0.2 µm Stericup®-GP filter (EMD Millipore Cat. No. SCGPU05RE); (3) Tris saline (TS): 20 mM Tris-Cl, 300 mM NaCl, pH 8; and (4) Native Lysis buffer (NB): 50 mM Na-phosphate, 300 mM NaCl, 10 mM imidazole, pH 8 (Boston Bioproducts Cat. No. BP-232). In each case, 5 mL buffer was added to each gram wet cell pellet. All buffers were supplemented with Protease Inhibitors Set VII (EMD Millipore Cat. No. 539138) at 1 mL/10 g wet cell pellet. For chemical lysis, cell pellets were resuspended in lysis buffer(s) and incubated at room temperature (RT) for 30 min. In certain cases, buffers were supplemented with rLysosyme™ solution (5 KU/gram wet cell pellet, EMD Millipore Cat. No. 71110-3) and Benzonase® nuclease (125 U/gram wet cell pellet, EMD Millipore Cat. No. 71205-3). For mechanical lysis, pellets were resuspended as described above then sonicated for 10 cycles on ice (1 cycle = 3 pulses) using a Branson Sonifier® Ultrasonic Cell Disrupter W-350 with microtip (60% duty cycle + 7 microtip limit, pulsed). In each case, insoluble cell debris was cleared by centrifugation at 9500 rpm for 20 min. Supernatant was removed, aliquoted, and stored at -20 °C.

Protein Detection and Quantitation
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) Test fractions were added to 1x NuPAGE® Sample buffer (Life Technologies Cat. No. NP0008) supplemented with dithiothreitol (DTT). Samples were mixed, denatured at 70 °C for 10 min., and loaded onto 4–12% gradient NuPAGE® Bis-Tris (MES) Gels (Life Technologies) with 5 µL of Mark12™ Standards (Life Technologies Cat. No. LC5677). Gels were run at 200 Volts for 40 min, stained/destained with SimplyBlue™ Safe Stain (Life Technologies Cat. No. LC6060) using the microwave method, and imaged. Bradford Assay Concentration for various protein fractions was determined using the Coomassie Plus Assay Kit (Thermo Scientific Cat. No. 23236) according to manufacturer’s instructions. Briefly, a set of protein standards was prepared using bovine serum albumin (BSA, range 0–1500 µg/mL). In a microplate, a 10 µL sample (or BSA standard) was added to 300 µL Coomassie reagent. The reactions were mixed for 1 min on a plate shaker then incubated at RT for 10 min. All samples were analyzed by detecting their absorbance at 595 nm (A595) and the concentration was determined using the derived BSA standard curve. IR-based Quantitation Proteins were quantitated using the Direct Detect™ assay free sample card and Direct Detect™ spectrometer. Each card contained four hydrophilic polytetrafluoroethylene (PTFE) membrane positions, each surrounded by a hydrophobic ring to retain analyzed sample within the device’s IR beam. All measurements were performed using 2 µL of sample per membrane position. A ”buffer only” sample was also analyzed as a reference blank. Sample concentration was determined in reference to a calibration method. Direct Detect™ quantitation only requires a standard curve to be generated once, at time of system installation. For all experiments, the system was calibrated using National Institute of Standards & Technology (NIST)-certified BSA SRM927d in phosphatebuffered saline (PBS). A series of ten concentration points from 0.125 mg/mL to 5 mg/mL was used to generate the instrument calibration curve.

6XHIS-CRP Affinity Purification using PureProteome™ Magnetic beads
For each reaction, a 50 µL slurry of PureProteome™ nickel magnetic beads (EMD Millipore Cat. No. LSKMAGH10) was initially washed with appropriate lysis buffer in a 1.5 mL tube. 1 mL lysate was then added to each bead fraction and reactions were incubated at RT with endover-end mixing for 30 min. Tubes were then placed on a PureProteome™ magnetic stand (EMD Millipore Cat. No. LSKMAGS08) to collect beads and remove the unbound fraction (flow through). Beads were washed 3X with 1 mL lysis buffer; wash fractions were pooled and saved. 6XHIS-CRP was eluted in 200 µL of Elution buffer (Boston Bioproducts Cat. No. BP-236). Two elution steps were performed.

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A. Cell Pellet Wet Mass Comparison
1.5 1x LB + IPTG O/N Media 1.0 1.355

Results
High recombinant protein yield is dependent not only on strategies for cell lysis and affinity purification, but also on the quality of the induction culture and level of protein expression within these cells. Traditional expression schemes involving pET systems require constant monitoring of the bacterial culture’s density to identify the optimal point at which induction, by addition of IPTG, can be initiated. After a short incubation (~ 4 h), cultures must then be harvested. Auto-inducible media, such as the Overnight Express™ systems, enable inductions to be carried on overnight without the need for tedious monitoring or IPTG addition. This method is based on differentially metabolizable medium components, resulting in high-density growth and automatic induction of lac -promoter-driven expression constructs. Figure 2 highlights a comparison of standard E. coli induction (LB + IPTG) to that using Overnight Express™ medium. The Overnight Express™ system resulted in ~ 3.5X greater cell pellet wet mass from the same starting volume. More significantly, the gel blots demonstrate that comparable amounts of total lysate and purified protein (6XHIS-CRP) were extracted from cell pellets of equivalent wet mass indicating that greater overall cell mass in the Overnight Express™ cultures correlates with increased protein yield per preparation.

Cell Pellet Wet Mass (g)

0.5

0.394

0.0

Induction Medium

B.
In

LB FT E1 In FT

O/N Exp E1 M kDa 200 116 97 66 55

6XHIS-CRP [26kDa]

36 31 22 14 6 In = Lysate E1 = Eluate #1 FT = Flowthrough M = MW Markers

Figure 2. Overnight Express™ medium yields same amount of product, but with automatic induction. (A) 500 mL bacterial cultures were induced by either traditional means or using Overnight Express™ medium. Following harvest, total cell pellet wet mass was determined. (B) An equivalent wet mass of cells from each induction method was lysed using BugBuster® reagent. 6XHIS-CRP was purified from the resulting lysates using PureProteome™ nickel magnetic beads. Yields were compared using SDS-PAGE.

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Once harvested, cells must be lysed under proper conditions to maximize extraction of soluble and functionally active protein. Through empirical testing, different detergent-based solutions composed of particular types and concentrations of detergents, buffers, salts and reducing agents have been developed to provide the best possible results for a particular protein or cell type. A myriad of commercial and homebrewed reagents, all varying in detergent makeup and buffering components, are available for E. coli cell lysis. Reagents, such as protease inhibitors or enzymes, can also be included to maximize extraction. One such agent, rLysozyme™ solution, contains a highly active enzyme that hydrolyzes structural N-acetylmuramide linkages, thereby degrading bacterial cell walls. A second component, Benzonase® nuclease, can be used to remove liberated DNA and RNA species. We compared the performance of BugBuster® protein extraction reagent to that of a homebrew lysis buffer by using SDS-PAGE and the Direct Detect™ IR-based quantitation system to measure protein yields (Figure 3A and 3B). The BugBuster® reagent (BB) liberated far more protein than the homebrew lysis buffer (HB). For both buffers, Benzonase® nuclease addition resulted in a slight increase in total lysate. Benzonase® nuclease significantly reduced solution viscosity, greatly simplifying subsequent sample processing (Figure 3C). rLysozyme™ had a dramatic effect on yield, suggesting that the critical step in the extraction process is complete bacterial lysis. Moreover, when used in combination with BugBuster® reagent, rLysozyme™ solution and Benzonase® nuclease appeared to have a greater than additive effect on total protein yield. Lastly, we found that protein quantitation using SDS-PAGE (Figure 3A) and Direct Detect™ IR-based quantitation (Figure 3B) yielded comparable results, suggesting that this benchtop spectrometry system provides a fast and accurate way of determining lysate concentration prior to freezing.

A.
rLysozyme™ Solution Benzonase® nuclease NI HB + HB - - + BB BB + BB + + BB M 200 116 97 66 6XHIS-CRP [26kDa] 55 36 31 22 14 6

NI = non-induced HB = Homebrew buffer

BB = BugBuster® M = MW Markers

B.
Protein Concentration (µg/mL)
10000 7500 5000 2500
HB = Homebrew BB = BugBuster®

0 rLysozyme™ Benzonase® Lysis Method

HB

+ HB

BB

+ BB

+ BB

+ + BB

C.
Without Benzonase® nuclease (gooey, viscous) With Benzonase® nuclease (not viscous, easy pipetting)

Figure 3. BugBuster® reagent is superior to “Homebrew” lysis buffer and BugBuster® reagent with both Benzonase® nuclease and rLysozyme™ solution yielded lysates with the most 6XHIS-CRP. (A) E. coli lysates (5 µL of 1 mL total lysate) from various lysis protocols were fractionated and analyzed by SDS-PAGE. A band corresponding to 6XHIS-CRP is prominently visualized in the BB +/+ lane. (B) Cleared cell lysates (2 µL of 1 mL total) were spotted on assay cards and quantified using the Direct Detect™ spectrometer. In each case, bars represent the average of 3 independent samples. (C) The images clearly demonstrate the difference in sample viscosity between Benzonase®-treated and untreated lysates.

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While chemical extraction methods are more common, mechanical methods, such as sonication, are still commonly employed for bacterial cell disruption. Although effective at liberating proteins, sonicated samples can have reduced yield of active protein due to denaturation or aggregation from localized over-heating. Sonicators, when compared to the cost of lysis buffers, are also quite expensive. To examine the efficiency of sonication, we compared total protein yield for sonication when used in conjunction with three typical buffer types to that of the BugBuster® chemical method (Figure 4). BugBuster® reagent, when used in conjunction with rLysozyme™ solution and Benzonase® nuclease, liberated for more protein than sonication. The purification of recombinant proteins has been greatly facilitated by the incorporation of affinity tags, such as polyhistidine, into the protein coding sequence. Affinity purification is further enhanced through the use of affinity ligands immobilized on paramagnetic beads. In these systems, the beads (and bound protein) are isolated using a magnet rather than by centrifugation, thereby greatly reducing the chance of including untagged, irrelevant proteins in the purified sample. We used PureProteome™ nickel magnetic beads to purify 6XHIS-CRP (Figure 5). The purity of the eluted fraction was verified by SDS-PAGE. PureProteome™ magnetic beads cleanly purified Histagged CRP from all of the various lysates tested. More importantly, the bead-based purification was compatible with BugBuster® protein extraction reagent as well as with rLysozyme™ solution and Benzonase® nuclease, thereby eliminating the need for dilution or buffer exchange. Protein concentration was determined by Direct Detect™ quantitation, which yielded concentration measurements equivalent to the Bradford assay (Figure 5B). Direct Detect™ quantitation also eliminated the tedium of reference sample preparation and standard curve analysis. Neither detergents nor reducing agents commonly found in bacterial lysis buffers affected the accuracy of Direct Detect™ quantitation.

Extraction Efficiency
1000

Protein Concentration (µg/mL)

7500

10 Sonication Cycles

5000

2500

0

Homebrew

Tris-NaCI

Native Buffer

BugBuster® (No sonication)

Lysis Buffer Figure 4. BugBuster® protein extraction reagent was more effective at liberating protein, even without sonication, than three other typical lysis buffers that were used with 10 cycles of sonication. Sonication-free cell lysis using BugBuster® reagent (plus Benzonase® and rLysozyme™) was compared to three typical lysis buffers (no Benzonase®/rLysozyme™) used in conjunction with sonication. Cleared cell lysates (2 µL of 1 mL total) were spotted on assay cards and quantified using the Direct Detect™ spectrometer. In each case, bars represent the average and standard deviation of 3 independent samples.

Addition of both rLysozyme™ solution and Benzonase® nuclease boosted protein recovery in BugBuster® Protein Extraction Reagent (lane 4). When only rLysozyme™ Solution was added (no Benzonase® nuclease addition), handling and processing was very difficult and is not recommended (Lane 6). Protein quantitation using a Bradford assay and the Direct Detect™ IR-based quantitation system (B) provided results in agreement with SDS-PAGE results (A).

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Summary
Optimizing key parameters for all steps of recombinant protein production, from cell culture, through sample purification, to final analysis, can dramatically increase protein yield, saving time and increasing the consistency of downstream structural and functional analyses. Overnight Express™ medium yielded significantly more total protein than the standard IPTG induction scheme with far fewer hands-on steps required. As a result, this system is ideally suited for screening experiments, in which parallel expression of multiple protein constructs requires higherthroughput processing of cultures. Optimized protein sample preparation reagents, specifically BugBuster® protein extraction reagent, Benzonase® nuclease and rLysozyme™ solution demonstrated here, provide superior extraction results that yield higher protein product recovery, especially when used in combination. Our results indicate that the combination of these three reagents offers superior performance to other standard lysis buffers, with or without sonication. By eliminating the need for sonication, this combination of reagents also minimizes risk of protein denaturation loss of specific activity. This reagent combination is also compatible with affinity purification using PureProteome™ magnetic beads and subsequent quantitation with the Direct Detect™ spectrometer. Structural studies and functional genomics, which involve the parallel production of multiple, diverse proteins, require tools enabling rapid, universal protein sample preparation and a protein quantitation method that is compatible with complex samples. Using the optimized reagents described in this study, together with benchtop IR-based protein quantitation as provided by the Direct Detect™ system, has the potential to improve the reproducibility, accuracy and throughput of proteomics research, finally bridging the gap between the genome and the proteome.

A. SDS-PAGE
M 2 3 4 HB HB BB -B +B +B -L -L +L - + - + 5 BB +B -L + 6 BB -B +L + + 7 BB -B -L

6XHIS-CRP

HB = Homebrew BB = BugBuster® Protein Extraction Reagent B = Benzonase® Nuclease L = rLysozyme™ Solution M = MW Marker NR = Not recommended

B.  Direct Detect™ IR-based Quantitation System and Bradford Assay Quantitation
800 HB = Homebrew BB = BugBuster® Reagent Direct Detect™ Bradford

Concentration (µg/mL)

600

400

200

0 rLysozyme™ Benzonase®

HB

+ HB

+ + BB

+ BB

+ BB

BB

Lysis Method Figure 5. PureProteome™ nickel magnetic beads cleanly purified the His-Tagged CRP from all of the various lysates. (A) Eluted fractions (5 µL of 400 µL total) from various lysis buffer preparations were fractionated and analyzed by SDS-PAGE to assess purity. (B) 6XHIS-CRP concentration was determined using both the Direct Detect™ IR-based quantitation system and Bradford assay.

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EMD Millipore, the M mark, Direct Detect, Overnight Express, PureProteome, and rLysozyme are trademarks of Merck KGaA, Darmstadt, Germany. Benzonase, BugBuster, Montage, and Stericup are registered trademarks of Merck KGaA, Darmstadt, Germany. Mark12 and SimplyBlue are trademarks and NuPAGE is a registered trademark of Invitrogen Corporation. Sonifier is a registered trademark of Branson Ultrasonics Corporation. Lit. No. AN3302EN00 03/2012 LS SBU-12-05898 Printed in the USA. ©2012 EMD Millipore Corporation, Billerica, MA 01821 USA. All rights reserved.

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