Constant cell disruption system

Manufactured by Constant Systems

Sourced in United Kingdom

The Constant cell disruption system is a laboratory equipment designed for the efficient disruption and lysis of cells. It utilizes a controlled and consistent mechanical force to break apart cellular structures, facilitating the extraction and purification of cellular contents such as proteins, nucleic acids, and other biomolecules. The device operates based on the principles of cell disruption, providing a reliable and reproducible method for sample preparation in various biological and biochemical applications.

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Lab products found in correlation

Hitrap, by Cytiva (3 mentions) Ni nta agarose column, by Qiagen (2 mentions) Hitrap heparin hp column, by GE Healthcare (2 mentions) Superdex 75 column, by GE Healthcare (2 mentions) Ni nta agarose resin, by Beyotime (1 mentions) Himac cp 100wx, by Hitachi (1 mentions) Protein quantification assay kit, by Macherey-Nagel (1 mentions) Protease inhibitor tablet, by Roche (1 mentions) Benzonase, by Merck Group (1 mentions) Lemo21 de3 competent e coli cells, by New England Biolabs (1 mentions) Sp sepharose column, by GE Healthcare (1 mentions) Superdex 200, by GE Healthcare (1 mentions) Gateway expression system, by Thermo Fisher Scientific (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions) Flag tag antibody, by Proteintech (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)

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Cell disruption system (3 citations) Cell disruption (2 citations)

13 protocols using constant cell disruption system

Optimized Gene Expression in E. coli

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The optimized gene sequence was synthesized by
the Beijing Genomics Institute with pUC57 (pUC57-ω-TAEn) between
the BamHI and HindIII restriction
sites. The purpose gene was connected to pETDuet-1 with the corresponding
sites to form the gene expression vector. The plasmids were transformed
into E. coli BL21, and the cultivated
strain was screened by colony PCR using primers pETUP1 (ATGCGTCCGGCGTAGA)
and T7-Terminator (ATGCGTCCGGCGTAGA).
The resulting strains
were cultured in the LB culture medium. When the OD₆₀₀ reached
about 0.6–0.8, ITPG (0.6 mM) was added. After 8 h of induced
expression at 30 °C, the cells were harvested at 5000 rpm for
5 min at 4 °C. Cell fragmentation was achieved by constant cell
disruption systems (Constant Systems) at 30 kpsi. The supernatant
was purified on a Ni-NTA agarose resin obtained from Beyotime Biotechnology.

Feng X., Guo J., Zhang R., Liu W., Cao Y., Xian M, & Liu H. (2020). An Aminotransferase from Enhydrobacter aerosaccus to Obtain Optically Pure β-Phenylalanine. ACS Omega, 5(14), 7745-7750.

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Recombinant Protein Localization in E. coli

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The recombinant strain with pET28a-INP-Faa was cultivated in LB broth containing 50 µg ml⁻¹ kanamycin at 37 ℃. IPTG was added at a final concentration of 0.2 mM for 4 h of induced expression at 30 ℃ when the OD₆₀₀ reached 0.6–0.8. The cells were harvested at 6000 rpm for 10 min at 4 ℃ and washed three times with Tris–HCL (pH 7.8, 50 mM).
To demonstrate location of INP-Faa, harvested cells were fractionated to obtain cytoplasm and membrane fractions according to the method proposed by Jochen [35 (link)]. The cells were resuspended in PBS buffer (pH 8.0, 50 mM) to set OD₆₀₀ as 5.0 and then crushed by constant cell disruption systems (Constant Systems) at 30 Kpsi. The suspension was centrifuged at 6,000 rpm for 10 min to remove undisrupted cells and large cell debris. The clarified extract was then centrifuged at 34,500 rpm for 1 h (Himac CP100WX, Hitachi, Japan) to obtain proteins from the periplasm and cytoplasm. The insoluble part was suspended in PBS containing MgCL₂ (0.01 mM) and Triton X-100 (2%) and incubated at 25 ℃ for 30 min to dissolving inner membrane, and then the resuspended components was centrifuged at 34,500 rpm for 1 h to get the outer membranes which was insoluble. The different fractionated samples were mixed with protein loading buffer and boiled for 2 min, then the mixture was determined by 10% SDS-PAGE. E. coli harboring pET28a-Faa was used as a reference.

Feng X., Jin M., Huang W., Liu W, & Xian M. (2021). Whole-cell catalysis by surface display of fluorinase on Escherichia coli using N-terminal domain of ice nucleation protein. Microbial Cell Factories, 20, 206.

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Crude Enzyme Extraction Protocols

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Crude enzyme extract from the fermentation media was prepared according to two different types of disruption protocols. Glass beads disruption protocol was applied as described above, except for the sediment from 100 µl of media, which was resuspended in 300 µl of Glass Bead Extraction Buffer; as an alternative, high pressure continual cell disrupter (Constant cell disruption systems, Constant Systems LTD, UK) was tested in parallel. Biomass was first centrifuged at 7000 g for 10 min at 4°C and then resuspended in potassium phosphate buffer (0.1 M, pH 8). Cell disruption took place at 4°C, in one cycle at 40 kPSI. The resulting lysate was ultracentrifuged at 50,000 g, 4°C for 30 minutes.

Markošová K., Camattari A., Rosenberg M., Glieder A., Turner N.J, & Rebroš M. (2018). Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris. Biotechnology letters, 40(1).

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Overexpression and Purification of rAgaB-4 from E. coli

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E. coli BL21(DE3)(pET-AgaB-4) cells were cultured in 1 L of LB broth containing kanamycin (30 μg/mL), with shaking at 37 °C. Cells were cultured to an OD₆₀₀ of 0.4–0.6. Subsequently, IPTG (0.1 mM) was added to induce rAgaB-4 expression at 20 °C for 24 h. Cells were harvested by centrifugation at 8000×g for 30 min and were then resuspended in lysis buffer. The cell suspension was disrupted using Constant Cell Disruption Systems (Constant Systems Ltd, Warwick, UK). The cell lysate was centrifuged at 8000×g for 15 min at 4 °C, and the resulting supernatant was filtered through a 0.22-μm membrane and applied to a 5-mL HiTrap™ excel affinity chromatography column (GE Healthcare, Uppsala, Sweden) according to the manufacture’s instruction. The purity of the eluted fusion protein was analyzed through 12.5% SDS-PAGE, and the protein concentration was determined using a Protein Quantification Assay Kit (MACHEREY–NAGEL, Düren, Germany).

Chen Z.W., Lin H.J., Huang W.C., Hsuan S.L., Lin J.H, & Wang J.P. (2018). Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4 from Paenibacillus agarexedens. AMB Express, 8, 49.

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Purification of PTMP1 from Cell Pellets

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The 5 ml lysis buffer (10 mM Tris-Cl, 100 mM sodium phosphate, pH 8.0) is used to resuspend the harvested cell pellets per gram wet weight. Through the use of constant cell disruption systems (Constant Systems), the samples were lysed at 20 kPsi before they were centrifuged for 20 min at 4 °C at 18,000 g. The debris of the cell was gathered for purification. To extract the PTMP1, the cell lysate pellet was resuspended in 10 % (v/v) acetic acid and the extracting solution was centrifuged for 20 min at 4 °C at 18,000 g. Using Spectra/Por molecular porous membrane tubing (MWCO 14,000 Da, Spectrum Laboratories, USA), the supernatant was collected and dialyzed at 4 °C in 1 % (v/v) acetic acid buffer overnight. Through freeze-drying process, the purified sample was concentrated, which will be used for further purification. The concentrated PTMP1 was injected to Aquapore RP-300 column (C8, 250 × 7.0 mm, Brownlee, Perkin-Elmer, USA) at the flow rate of 1.0 mL/min and following the same gradient condition as the previous PTMP1 purification method from mussels [15 (link)].

Yoo H.Y., Song Y.H., Foo M., Seo E., Hwang D.S, & Seo J.H. (2016). Recombinant mussel proximal thread matrix protein promotes osteoblast cell adhesion and proliferation. BMC Biotechnology, 16, 16.

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E. coli Membrane Preparation Protocol

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E. coli membrane preparation was performed as described with minor modifications^{29 (link)}. The cell pellet was thoroughly resuspended in 10 mL of 65 mM MOPS buffer pH 7.5 per gram of cells supplemented with protease inhibitor tablets (Roche; 1 tablet per 50 mL), 2.5 U mL⁻¹ of benzonase (Sigma) and 100 mM MgSO₄. The cells were disrupted using two passes at 30 kPa through a pre-cooled Constant cell disruption system (Constant Systems Ltd). The lysate was centrifuged for 15 min at 16,000 rcf, and the pelleted unbroken cells and large cell debris were discarded. The supernatant was centrifuged for 90 min at 200,000 rcf to collect the fragmented membranes. After centrifugation the supernatant was discarded and the membrane pellets were thoroughly resuspended with 65 mM MOPS pH 7.5 with the aid of a Dounce homogenizer.

Barsottini M.R., Copsey A., Young L., Baroni R.M., Cordeiro A.T., Pereira G.A, & Moore A.L. (2020). Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen Moniliophthora perniciosa. Communications Biology, 3, 263.

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Purification of Recombinant HsIP6K1

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The cDNA of HsIP6K1 (residues 9 to 441) was subcloned into the pDest-566 vector. This vector encodes a 6 × His tag, a maltose binding protein tag and TEV protease cleavage site at the N-terminus. Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid. An overnight culture of the transformed E. coli cells was inoculated into nutrient-rich 2xYT medium supplemented with 0.6 mM l-rhamnose at pH 7.5 and grown at 37 °C to A₅₉₅ = 0.7. Isopropyl β-d-thiogalactopyranoside (0.1 mM) was then added and cultures were continued at 15 °C for 2 days. The cells were disrupted using a constant cell disruption system (Constant Systems Ltd., Northants, UK) under 20 KPsi. Recombinant HsIP6K1 was purified with a Ni-NTA agarose column (Qiagen, Germantown, MD, USA) followed by a HiTrap™ Heparin HP column (GE Healthcare, Pittsburgh, USA). The purity was estimated to be > 80% as judged by SDS-PAGE. The purified proteins were concentrated and stored at − 80 °C.

Rajasekaran S.S., Illies C., Shears S.B., Wang H., Ayala T.S., Martins J.O., Daré E., Berggren P.O, & Barker C.J. (2018). Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity. Cellular Signalling, 42, 127-133.

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Recombinant Lysostaphin SH3b Expression

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A DNA fragment encoding residues 402–493 of lysostaphin was cloned into a pET15b expression vector using NcoI and XhoI restriction sites. The protein was overexpressed in E. coli BL21(DE3) in LB medium by induction with 0.1 mM IPTG for 6 h in 25 °C. The cell pellet was suspended in 20 mM Tris-HCl pH 7.0, 1 M NaCl, 10% glycerol and disrupted with a Constant Cell Disruption System (Constant Systems Ltd.). The cell lysate was dialyzed against 20 mM Tris-HCl, pH 7.0, 50 mM NaCl. SH3b was purified by ion exchange chromatography on WB40S resin (Bio-Works, Uppsala, Sweden), eluted with about 0.5 M NaCl in 20 mM Tris buffer pH 7.0, followed by gel filtration on Superdex 75 column (GE Healthcare) in 20 mM Tris-HCl, pH 7.0, 50 mM NaCl buffer.

Mitkowski P., Jagielska E., Nowak E., Bujnicki J.M., Stefaniak F., Niedziałek D., Bochtler M, & Sabała I. (2019). Structural bases of peptidoglycan recognition by lysostaphin SH3b domain. Scientific Reports, 9, 5965.

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Purification of Lysostaphin Enzymes

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E. coli cell pellet containing overexpressed enzymatically active domain of lysostaphin (Lss_EAD), enzymatically active domain of LytM (LytM_EAD), mature lysostaphin (Lss), or lysostaphin cell wall binding domain (Lss_CBD) was suspended in 20 mM Tris-HCl, pH 7.0, 1 M NaCl, and 10% glycerol (50 mM NaCl for LytM_EAD) and disrupted by Constant Cell Disruption System (Constant Systems Ltd.). Cell lysate was dialyzed against 20 mM Tris-HCl, pH 7.0, 50 mM NaCl, and purified by exchange chromatography in NaCl gradient (SP Sepharose column; GE Healthcare). Further purification was done by gel filtration on Superdex 75 column (GE Healthcare) in 20 mM Tris-HCl, pH 7.0, 200 mM NaCl, and 10% glycerol buffer. Overexpressed Chimera and revChimera enzymes were purified as described above with one exception; E. coli were lysed by sonication.

Jagielska E., Chojnacka O, & Sabała I. (2016). LytM Fusion with SH3b-Like Domain Expands Its Activity to Physiological Conditions. Microbial Drug Resistance, 22(6), 461-469.

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Expression and Purification of Recombinant Inositol Kinases

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Recombinant human IP6K2 and the human IPMK and PPIP5K2 kinase domain were prepared as previously described.^{41 (link),47 ,48 (link)} Human IP3KA kinase domain (residues 173–461, UniProtKB code P23677) was cloned into the pDest-566 vector by the Gateway expression system (Invitrogen).^{48 (link)} Genscript synthesized the codon-optimized cDNAs for expression in Escherichia coli of 6xHis-MBP tagged human IP6K1 (N-terminally tagged with 6×-His followed by MBP) and IP6K3 (N-terminally tagged with 6×-His followed by Sumo). The procedures for IP6K1, IP6K3, IPMK, and IP3KA expression and purification were identical to those used for IP6K2 except that the MBP tag was cut by TEV protease in IPMK and IP3KA. The cells were disrupted using a constant cell disruption system (Constant Systems) under 20 kpsi. Recombinant IP6Ks were purified with a Ni-NTA agarose column (Qiagen) followed by a HiTrap heparin HP column (GE Healthcare). As a final step, a Superdex 200 gel filtration column (GE Healthcare) was used with a running buffer of 150 mM NaCl and 20 mM Tris-HCl, pH 7.5. The purity of these proteins was estimated to be >80% as judged by SDS-PAGE. The purified proteins were stored in aliquots at −80 °C.

Zhou Y., Mukherjee S., Huang D., Chakraborty M., Gu C., Zong G., Stashko M.A., Pearce K.H., Shears S.B., Chakraborty A., Wang H, & Wang X. (2022). Development of Novel IP6K Inhibitors for the Treatment of Obesity and Obesity-Induced Metabolic Dysfunctions. Journal of medicinal chemistry, 65(9), 6869-6887.

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