Improved, All-in-one B-PER Reagent for Bacterial Protein Extraction - DE (2024)

B-PER Complete Reagent provides gentle, efficient, and cost effective protein extraction fromE. coliand other bacterial cells

byEglė Namavičiūtė, M.S.1; Kristina Pagarauskaitė, M.S.1; Kęstutis Bargaila, M.S.1; Juozas Šiurkus, Ph.D.1- 02/19/14

Gram-positiveBacilli,Lactobacilliand Gram-negativeE. coliare 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 asE. colithere 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-positiveCocciexhibit greater mechanical resistance than rod-shapedBacilli.

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

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) andWELQut(# EO0861) proteases. During this experiment, we expressed and processed 6xHis-[WELQut recognition site]-Klenow fragment and GST-[HRV3C recognition site]-Syk proteins inE. coliER2566,as 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).

Conclusions

Methods

Cells cultivation and protein synthesis:

For expression of recombinant proteins,E. colicells 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 5000xgfor 10 minutes. Cell pellets were either used fresh or stored at -20^oC 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 16,000xgfor 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 usingWELQut 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).