Gram-positive BacilliLactobacilli and Gram-negative E. coli are favorite recombinant protein production systems due to their simple cultivation and easy genetic manipulation. In these prokaryotic recombinant cell factories, the accumulation of target product can be secreted into the cultivation media or accumulated inside of the cell. In Gram-negative bacteria such as E. coli there is an alternative intracellular accumulation option in which the target protein is delivered to the periplasmic space.

The deposition of desired products in the intracellular cavity is often preferred due to the possibility of obtaining cell paste harbouring target product in a very condensed form via centrifugation or filtration. In addition, prokaryotic cell cultivation techniques allow production of desired bio-products at high cell densities. Therefore, much higher total yields of the recombinant protein can be obtained from less cultivation volume, compared to bioprocesses with extracellular product accumulation. The recovery of intracellular products is achieved by introducing a cell disruption/lysis step following cell harvest.

The cell envelope structure of Gram-positive and Gram-negative bacteria types are very different. The envelope of Gram-positive bacteria cells consists of a single peptidoglycan layer associated with teichoic acids. The cell envelope of Gram-negative bacteria is composed of three layers, including the lipopolysaccharide-rich outer membrane, a peptidoglycan layer, and the inner membrane of phospholipids which contains integral and peripheral proteins. The mechanical resistance of both types of bacteria is determined by the peptidoglycan layer. The single layer of peptidoglycan in Gram-positive bacteria cells is species-dependent and can be 2- to 20-fold thicker than in Gram-negative bacteria. Therefore, Gram-positive cells exhibit much greater structural strength. Furthermore, the mechanical strength of bacterial cells is also dependent on cell shape; spherical Gram-positive Cocci exhibit greater mechanical resistance than rod-shaped Bacilli.

The method for bacterial disruption is usually selected by taking into account features of the target product such as pH-sensitivity, thermostability, and resistance to detergents and chaotropes. Cell-disruption methods are made cost-effective by taking into account the process scale, available equipment and the cell type/mechanical features. The ideal protocol will have a minimal impact on the biochemical parameters of the target product, be compatible with downstream processing, and require minimal pre-treatment of crude material prior to isolation of the target product.

Currently, bacterial cell disruption methods can be classified as either mechanical or non-mechanical. Mechanical disruption of bacterial cells requires costly special equipment, where cells in suspension or semi-solid paste are lysed using high pressure, intensive mechanical agitation or grinding. Non-mechanical disruption can be subdivided into chemical, physical, or biological methods.  Chemical methods utilize alkaline or acidic reagents, detergents, chaotropic agents, or antibiotics. Physical methods involve exposing bacterial cells to ultrasound cavitations, osmotic shock, gas decompression, hydrodynamic cavitations, and/or freeze-thaw cycles.  Biological methods utilize various cell lysis enzymes, autolysis induction, phages and other biological factors which decompose the envelope of the cell.

Thermo Scientific B-PER Complete Bacterial Protein Extraction Reagent combines enzymatic and chemical components, allowing efficient but mild extraction of soluble proteins and isolation of inclusion bodies from frozen and fresh Gram-positive and negative cells. Unlike classic formulations of B-PER Reagent (Part No. 78248), the new B-PER Complete Reagent is a Tris-buffered detergent solution that contains lysozyme and Pierce Universal Nuclease; the all-in-one solution is stable for long-term storage at 4°C. The reagent is suitable for cost-effective laboratory scale cell lysis with low viscosity, thus eliminating the need for mechanical disruption. In this article, we present data to demonstrate the performance of the new B-PER Complete reagent.

Results and discussion

First, we evaluated performance of B-PER Complete Reagent to lyse and extract protein from Gram-negative E. coli ER2566 pLATE516xHis-Klenow and Gram-positive Bacillus subtilis strain WB800. We tested disruption efficiency with fresh (just after cultivation) and frozen cells. We differentiated and separately analyzed by gel electrophoresis both the total protein fraction (total sample immediately after incubation in reagent) and the soluble protein fraction (clarified extract after removal of cell debris by high speed centrifugation). We also examined lysed cells by light microscopy upon fuchsin staining. B-PER Complete Reagent efficiently lyses frozen and fresh cells of Gram-negative E. coli and Gram-positive B. subtilis constructs (Figure 1). For Gram-negative E. coli, significantly better yield resulted with frozen cells; therefore, we recommend using at least one freeze-thaw cycle before lysing E. coli. For fresh Gram-negative E. coli, we determined that the addition of 1mM EDTA to B-PER Complete is required (comparison data not shown).

Figure 1. Effective cell lysis of Gram-negative and Gram-positive bacteria with B-PER Complete Reagent. Fresh or frozen cells of E. coli and Bacillus were lysed in reagent, and then protein fractions were separated by SDS-PAGE (10% gels) alongside a protein ladder (Part No. 26616). Total (T) protein fraction is the initial lysate before removal of cell debris by centrifugation; the soluble (S) protein fraction is the final clarified lysate. Micrographs below are the corresponding fuschin-stained samples of cells after lysis.

Next we performed similar experiments to evaluate the lysis of E. coli ER2566cells expressing the following fusion proteins:  6xHis-Klenow (exo-) (fragment of E. coli DNA polymerase I, 67kDa), GST-StpA (S. aureus staphostatin A, 12kDa) and GST-Syk (human spleen tyrosine kinase, 29kDa). We compared results with B-PER Complete Reagent to those obtained using another popular lysis reagent: BugBuster™ Master Mix (EMD Millipore™ Corporation, #71456-4). After cell lysis and separation of cell debris, soluble protein yield was measured using Pierce BCA Protein Assay Kit (Part No. 23225) and then separated by SDS-PAGE. B-PER Complete performed similarly to BugBuster Master Mix when lysing frozen Gram-negative E. coli expressing recombinant protein (Figure 2). However, repeated experiments with Gram-positive cells revealed that BugBuster Master Mix reagent failed to lyse fresh or frozen Bacillus cells, whereas B-PER Complete Reagent effectively lysed both (Figure 3).

Figure 2. Similar performance of two commercial reagents for lysis of Gram-negative E. coli expressing various recombinant proteins. Pellets (0.5g) of frozen cells expressing three different fusion proteins (6xHis-Klenow, GST-StpA and GST-Syk) were disrupted using 2.5mL aliquots of B-PER Complete Reagent (left) or BugBuster™ Master Mix (right, EMD Millipore™ Corporation). Panels are stained 12% gels (SDS-PAGE) of control (C ), total (T) and soluble (S) protein fractions obtained by each protein extraction reagent. Unlabeled lanes are a protein ladder (Part No. 26616). Protein yields (concentrations) for soluble fractions were determined using the Pierce BCA Protein Assay Kit (Part No. 23225). C = control (E. coli lysate from before induction with IPTG); T = total protein (initial lysate before removal of cell debris by centrifugation); S = soluble protein (final clarified lysate of IPTG-induced culture).
Figure 3. B-PER Complete Reagent outperforms BugBuster™ Master Mix in lysis of Gram-positive bacteria. Panels are stained 12% gels (SDS-PAGE) of lysates from two separate sets of experiments to extract proteins from fresh and frozen Gram-positive B. subtilis WB800 bacteria cells with B-PER Complete Reagent (B-PER) or BugBuster Master Mix (Bug). Labeled lanes are total (T) and soluble (S) and insoluble (I) protein fractions, respectively. Unlabeled lanes are a protein ladder (Part No. 26616).

Lysis methods ultimately need to be compatible with downstream applications, such as isolation of purified target protein. We evaluated the compatibility of B-PER Complete with affinity purification of N-terminal His-tagged Klenow fragment using HisPur Ni-NTA Resin (Part No. 88222), and N-terminal GST-tagged StpA and Syk using Pierce Glutathione Agarose (Part No. 16101). All recombinant proteins were expressed in ER2566 E. coli strain under the same cultivation conditions. After recombinant expression, the cells were lysed with either B-PER Complete Reagent or BugBuster Master Mix reagents. Soluble protein fractions were separated by centrifugation, supplemented with required additives for Ni-NTA or GSH based purifications, and directly applied to the corresponding resin using batch chromatography. B-PER Complete Reagent is compatible with Ni-NTA purifications, including with the recommended addition of 1mM EDTA (Figure 4). However, B-PER Complete Reagent shows a clear advantage for GST-based purifications. Yields of purified GST-StpA and GST-Syk were significantly lower with lysates from BugBuster Master Mix vs. B-PER Complete Reagent (Figure 5). Apparently, components of BugBuster Master Mix negatively affected the biochemical interaction of the GST tag with immobilized GSH ligand.

Figure 4. EDTA is compatible with B-PER Complete protein extraction for Ni-NTA purification. Frozen (Fro) and fresh (Fre) E. coli cells expressing His-tagged Klenow were extracted using B-PER Complete Reagent containing no EDTA (-) or 1mM EDTA (+). HisPur Ni-NTA Resin (Part No. 88221) was used to purify the His-tagged protein. Stained 12% gel (SDS-PAGE) shows that 1mM EDTA required for fresh biomass disruption does not adversely affect His-tagged protein recovery by Ni-NTA.
Figure 5. B-PER Complete is compatible with both Ni-NTA and GSH affinity purification. Frozen cells expressing three different fusion proteins were lysed using B-PER Complete Reagent (B-PER) or BugBuster™ Master Mix (Bug) and then purified via Ni-NTA or glutathione agarose. Lanes are soluble protein fractions (S) and final elution fractions (E) from each purification resin. Control lane (C) is the total protein fraction of non-induced ER2566 cells. Unlabeled lanes are protein ladder (Part No. 26616). Arrows indicate pairs of results from respective lysis reagents; B-PER Complete provided significantly better results with GST-tagged proteins (glutathione purification).

Some applications require the removal of affinity tags from the purified protein using specific proteases. We determined whether B-PER Complete affects enzymatic activities of HRV 3C (Part No. 88946) and WELQut (# EO0861) proteases.  During this experiment, we expressed and processed 6xHis-[WELQut recognition site]-Klenow fragment and GST-[HRV3C recognition site]-Syk proteins in E. coli ER2566as previously described. For purification and on-column cleavage, lysates were incubated with the appropriate chromatography resin in batch mode, washed, and subjected to cleavage with adsorbed 6xHis-WELQut or GST-HRV3C proteases. After proteolytic digestion, flow-through and elution fractions were analyzed for the presence of cleaved target protein. Gel analysis revealed that B-PER Complete Reagent was fully compatible with this purification process (Figure 6); the reagent did not adversely affect the activity of either protease in this on-column digestion application. Lysates obtained using BugBuster Master Mix were also apparently compatible with these protease methods; however, yield was negligible in the GST-HRV3C system (Figure 6, lower right). We assume that this is because so little GST-tagged Syk was bound and available for on-column digestion, a consequence of the reagent’s incompatibility with glutathione-based purification (see Figure 5).

Figure 6. B-PER Complete is compatible with downstream processing by proteases. On-column cleavage of His and GST affinity tags by WELQut and HRV 3C proteases (above and below, respectively) enables recovery of purified, tag-free recombinant proteins (paired arrows to FT lanes). Lanes are soluble protein fractions (S), flow-through (FT) after incubation with protease, and final elution fractions (E) to strip columns of tags and tagged-proteases. Control lane (C) is the total protein fraction of non-induced ER2566 cells. Unlabeled lanes are protein ladder (Part No. 26616). Lysates prepared with B-PER Complete (B-PER) performed well in both systems, while BugBuster™ Master Mix (Bug) performed poorly in the GST-tagged system with HRV 3C protease.

B-PER Complete Reagent contains a unique non-ionic detergent and optimal concentrations of enzymes that aid in the efficient lysis and extraction of proteins. Lysates generated from Gram-positive and Gram-negative bacteria are directly compatible with protein estimation assays, affinity chromatography, and downstream processing by enzymes. B-PER Complete Reagent provides researchers with a valuable tool for the efficient and cost-effective lysis of a wide range of bacteria without the need for mechanical disruption.


Cells cultivation and protein synthesis:

For expression of recombinant proteins, E. coli cells were cultivated in LB medium containing 100µg/mL Ampicillin, at 37°C with shaking at 220 rpm. The induction of recombinant protein synthesis was performed at OD600 of 0.6-0.8 with 0.1mM IPTG. The recombinant synthesis was continued for 3 hours at 25°C with 220-rpm rotation.  For protein analysis of non-induced cells, a control sample was taken 10 minutes prior to synthesis induction.

Cell lysis:

Cell lysis was performed using B-PER Complete Bacterial Protein Extraction Reagent (Part No. 89821) and BugBuster Master Mix (EMD Millipore Corporation, #71456-4) according to manufacturer protocols. Briefly, bacterial cells in LB media were centrifuged at 5000xg for 10 minutes. Cell pellets were either used fresh or stored at -20oC before processing. For fresh Gram-negative cells, B-PER Complete Reagent was supplemented with 1mM EDTA final concentration. Cells were resuspended in 5mL B-PER Complete Reagent or BugBuster Master Mix per gram of wet mass and incubated for 15 minutes at room temperature on a shaking platform at slow speed. Following lysis, cell lysates were cleared by centrifugation at 16,000xg for 20 minutes at 4°C. Equal volumes (0.5µL) of total and soluble protein fractions were denatured in SDS sample buffer and separated using 12% SDS-PAGE.

Fuchsin staining of cells after lysis:

Samples were fixed on a glass slide using the heat method after 15-minute lysis at room temperature and stained with fuchsin for 1 minute followed by washing with distilled water. Images were obtained using an Olympus BX50 microscope with immersion objective and Olympus DP11 digital camera (total zoom x 1250).

Determination of protein concentration:

Protein concentration and yield was determined using the Pierce BCA Protein Assay Kit (Part No. 23225) and Bovine Serum Albumin Standards (Part No. 23208), according to the microplate procedure protocol.

Ni-NTA purification of 6xHis-Klenow and cleavage with 6xHis-WELQut Protease:

For purification of His-tagged Klenow using the batch method, 0.1mL of equilibrated HisPur Ni-NTA Resin (Part No.88222) was added to 0.5mL of lysate with 10mM imidazole final concentration added. The resin and lysate was incubated for 30 minutes at room temperature with end-over-end mixing. Unbound protein was removed by centrifugation, and the column was washed twice with wash buffer (100mM Tris-HCl pH 8.0, 500mM NaCl, 20mM imidazole). 6xHis-tagged Klenow protein was  specifically cleaved using WELQut Protease (# EO0861) according to manufacturer protocol. Uncut tagged protein was eluted using two column-volumes of elution buffer (100mM Tris-HCl pH 8.0, 500mM NaCl, 500mM imidazole).

Procedure for purification of GST-tagged Syk protein by batch method:

For purification of GST-Syk or GST-StpA using the batch method, 0.1mL of equilibrated Pierce Glutathione Agarose (Part No. 16101) was added to 0.5mL of lysate. The mixture was incubated for 60 minutes at room temperature with end-over-end mixing. Unbound protein was removed by centrifugation, and the column was washed twice with wash buffer (50mM Tris-HCl pH 8.0, 150mM NaCl) twice. GST-tagged Syk protein was specifically cleaved using HRV-3C protease (Part No. 88946) according to manufacturer protocol. Uncut tagged protein was eluted using two column-volumes of elution buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 10mM reduced L-glutathione).