Glass Bead Milling Research Articles

Evaluation and Optimization of Protein Extraction From E. coli by Electroporation.

Growing diversity of protein-based technologies dictates further development of bio manufacturing to lower the cost of production and maximize yields. Intracellularly expressed recombinant proteins must be extracted from production host prior to purification. Use of electroporation to obtain proteins from bacteria and yeasts has been demonstrated in several studies for different modes of operation and formats. Here we tested various protocols for protein extraction from Escherichia coli by means of electroporation. The tested protocols were compared to established extraction methods of ultrasonication and glass-bead milling in terms of protein yields and content of impurities such as host cell DNA and endotoxins in the lysate. Protein extraction yield was maximal when exponentially growing bacteria were treated at 37°C, regardless of the electroporation mode of operation (batch or flow). We were unable to eliminate co-extraction of host DNA and endotoxins, but with 8 × 1 ms, 5 kV/cm, 1 Hz pulses they were minimized. Yields with optimized electroporation (up to 86 g protein/kg dry weight) were inferior to those in ultrasonication (up to 144 g protein/kg dry weight) and glass-bead milling (up to 280 g protein/kg dry weight). Nevertheless, electroporation largely avoids cell lysis and disintegration with which the extract is a mix of extracted proteins with debris of the bacterial envelope and bacterial DNA, which necessitates further purification.

Spent brewer's yeast as a source of high added value molecules: a systematic review on its characteristics, processing and potential applications.

Development of new strategies to add-value to agro-industrial by-products are of environmental and economical importance. Innovative and low-cost sources of protein and bioactive peptides have been explored worldwide. Spent brewer's yeast (SBY) is the second most relevant by-product from the brewing industry, and despite its nutritional (about 50% protein, dry weight) and technological potential, it is still underused or needs to be disposed of. SBY cells need to be disrupted to release intracellular and cell wall proteins. This procedure has been performed using autolysis, glass bead milling, enzymatic hydrolysis and ultrasound processing. Enzymatic treatment is usually performed without prior purification and is a challenging process, which involves multiple factors, but has been successfully used as a strategy to add value to agro-industrial by-products. Scope and approach: in this review, we particularly focused on enzymatic hydrolysis as a strategy to promote SBY valorisation, illustrating the state-of-the-art processes used to produce protein extracts from this material as well as exploring fundamental concepts related to the particularities of yeast cell disruption and protein hydrolysis. Furthermore, innovative applications of value-added yeast by-products in food, biotechnological and pharmaceutical industries are presented and discussed. Key findings and conclusions: the discovery of valuable compounds found in spent yeasts as well as the development of new processing methodologies have been widening the possibilities of reuse and transformation of SBY as an ingredient and innovative matrix. Once released, yeast proteins and peptides may be applied as an innovative non-animal protein source or a functional and bioactive ingredient.

The One Hour Yeast Proteome

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 (x̄) peptide spectral matches and 34,255 (x̄) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

Large-Scale Extraction of Recombinant Proteins from Bacteria

Bacteria are particularly convenient for producing recombinant proteins for purification purposes. Suitable extraction methods for bacterial cells include sonication, glass bead milling, grinding with alumina or sand, high-pressure shearing using the French pressure cell (French Press), and lysozyme treatment. These procedures are applicable for preparing extracts from a variety of gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae, and gram-positive bacteria such as Bacillus subtilis. Disruption of bacterial cells by enzymatic means is commonly used because a relatively uniform treatment is obtained when cells are in suspension. This protocol describes the enzymatic disruption of E. coli.

A study of cell disruption of Candida mogii by glass bead mill for the recovery of xylose reductase

The release of xylose reductase (XR) from Candida mogii by cell disruption in a glass beads mill was studied using an experimental design. Statistical analysis of the results indicated that XR volumetric activity increases by using lower glass beads diameter and cell concentration, and by increasing the number of agitation pulses. Based on results attained in experimental design, assays were carried out aiming at the maximization of XR release. Under optimized conditions (300 μm glass beads, 45 g/l of cell concentration and 50 pulses), the XR volumetric activity reach 0.683 U/ml. Disruption with glass beads showed to be the most efficient method for XR release when compared to sonication process. The highest specific activity (0.175 U/mg of protein) was found in extracts obtained by suspension freezing and thawing, which suggests that this method can be used as a selective process of cell disruption for XR release.

Cloning, expression, purification, and characterization of a designer protein with repetitive sequences

An artificial protein containing alternating hydrophilic–hydrophobic blocks of amino acids was designed in order to mimic the structure of synthetic multiblock copolymers. The hydrophobic block consisted of the six amino acids Ala Ile Leu Leu Ile Ile (AILLII) and the hydrophilic block of the eight amino acids Thr Ser Glu Asp Asp Asn Asn Gln (TSEDDNNQ). The coding DNA sequence of the cluster was inserted into an commercial pET 30a(+) vector using a two step strategy. The expression of the artificial protein in Escherichia coli was optimized using a temperature shift strategy. Only at cultivation temperature of 24 °C after induction expression was observed, whereas at 30 and 37 °C no target protein could be detected. Cells obtained from a 15 L bioreactor cultivation of E. coli were disintegrated by mechanical methods. Interestingly, glass bead milling and high pressure homogenization resulted in a different solubility of the target protein. The further purification was carried out by affinity chromatography using the soluble homogenized protein. Extreme conditions (6 M urea, 0.5 M NaCl) were applied in order to prevent aggregation to insoluble particles. The designer protein showed an extremely high tendency to form dimers or trimers caused by intermolecular interactions which were even not broken under the conditions of SDS–polyacrylamide gel electrophoresis, rendering the behavior during purification different from proteins usually found in nature. The protein preparation was not completely pure according to SDS–PAGE stained by Coomassie blue or silver. In MALDI-TOF-MS, nano-ESI qTOF-MS of the entire protein preparation and nano-ESI-MS after digestion by trypsin and chymotrypsin impurities were not detectable.

A Comparison of Cell Wall Disruption Techniques for the Isolation of Intracellular Metabolites from Pleurotus and Lepista sp.

Different techniques were compared for their effectiveness in the disruption of the rigid cell walls of Basidiomycetes. Grinding under liquid nitrogen, stirred glass bead milling and enzymatic cell lysis were applied to the mycelia of Pleurotus sapidus and Lepista irina grown submerged. Each of the disruption procedures was evaluated by testing the quantity and quality of released intracellular metabolites: DNA, RNA, enzymes, and secondary metabolites. The most suitable method for nucleic acid isolation was grinding under liquid nitrogen, while bead mill homogenization was the superior technique for isolation of active enzymes. A new effective method is proposed for isolation of secondary metabolites with the aid of bead milling of fungal mycelia.

Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis

Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, Cladosporium herbarum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid nitrogen, grinding at ambient temperature, sonication, glass bead milling and freeze–thawing. DNA purification and recovery from the lysates were performed using a commercially available system based on the selective binding of nucleic acids to glass milk. A simple quantitative competitive polymerase chain reaction (QC-PCR) assay was developed for use in determining copy numbers of the internal transcribed spacer (ITS) regions of the ribosomal RNA operon (rDNA) in the total DNA extracts. These quantitative analyses demonstrated that the method using glass bead milling was most effective in recovering PCR templates from each of the different types of conidia both in terms of absolute copy numbers recovered and also in terms of lowest extract to extract variability. Calculations of average template copy yield per conidium in this study indicate that the bead milling method is sufficient to support the detection of less than ten conidia of each of the different organisms in a PCR assay.

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1:1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola.

Isolation and characterization of recombinant annexin V expressed in Saccharomyces cerevisiae

The cloning, purification and characterization of full-length annexin V, expressed intracellularly in Saccharomyces cerevisiae is detailed. Following homogenization in a glass bead mill, clarification by ultracentrifugation and fractional ammonium sulfate precipitation, the 319 amino acid protein was purified by column chromotography on phenyl-Sepharose and heparin-Sepharose. Annexin V elutes on reverse phase C 4 silica as a single peak with greater than 97% homogeneity and is further characterized by a molecular mass of 34 kDa from electrophoresis under reducing conditions on SDS gels. Dynamic light scattering experiments reveal annexin V exists as a monomer in solution. Amino terminal Edman degradation afforded no sequence, therefore the carbamidomethylated protein was chemically cleaved with cyanogen bromide. Separation of the resulting peptide fragments on reverse phase HPLC followed by N-terminal sequencing and electrospray mass spectrometry supported the correct sequence as well as the existence of an acetyl blocking group on the N-terminus. The protein exhibits an isoelectric point of 4.73 by column chromatofocusing. Secondary structure predictions from CD spectroscopy indicate that the molecule is correctly folded. In anticoagulant assays, the purified protein exhibits dose-response effects in activated partial thromboplastin time (APTT) prolongation and doubles the clotting time of control human plasma at 70 μg ml −1. More specifically, in a factor Xa inhibition assay in which the activation of factor X via the tissue factor-factor VIIa complex is monitored by the cleavage of a factor Xa chromogenic substrate, recombinant annexin V exhibits a 50% inhibitory concentration (IC 50) in the low nanomolar range.

Disruption of a filamentous fungal organism (N. sitophila) using a bead mill of novel design I. General disruption characteristics

The disruption of a typical filamentous fungus, a native strain of Neurospora sitophila, was studied using a glass bead mill of novel design (the Sulzer Annu Mill 01). Cell concentration (in the range of 2.5–5 g dry weight/L) had little influence on the disruption attained. Disruption increased with increasing rotor speed (1000 –4000 r.p.m.) and number of passes (up to six passes) through the Annu Mill. Disruption was observed to follow traditional first-order kinetics for bead mills possessing predominantly plug flow characteristics. It was concluded that in general the Annu Mill would be applicable for the disruption of filamentous organisms.

Extraction of chromatophores from Rhodospirillum rubrum in aqueous two-phase systems: A method for large-scale isolation of membrane particles

Chromatophores, membrane vesicles with the capacity of cyclic photophosphorylation, have been isolated from Rhodospirillum rubrum cells on a pilot plant scale. Results of disintegration in a glass bead mill and in a high pressure homogenizer were compared. The chromatophores were isolated from the crude extract by extraction in aqueous two-phase systems. In systems of polyethylene glycol (PEG) and dextran the chromatophores were partitioned to the upper PEG phase by the addition of PEG-palmitate. Most of the proteins and nucleic acids were forced to the bottom phase by addition of sodium chloride. Methods to prevent precipitation of the chromatophores were studied.