微生物培养基质量控制技术和标准

微生物培养基的酸碱度、凝胶强度和选择性等直接影响到培养基的质量,在理化试验方法中采用连接可渗透陶器型液体接头的电极和平头电极或者连接微型探头的电极可分别测定液体和固体培养基的pH值,而采用Gelometer和the LFRA Texture Analyser可测定固体培养基的凝胶强度。在微生物学方法中固体培养基采用倾注平板法、涂布法、划线法（半定量法）、改良的Miles-Misra法等测定生长情况,液体培养基采用稀释法测定生长率,用目标菌和杂菌的混合菌株评价选择性增菌培养基的选择性,利用OD值评价液体培养基生长率等。ICFMH（国际食品微生物学和卫生学委员会培养基工作组）、ISO、FDA以及我国卫生部等相继制定了培养基质量控制的标准,但目前还没有一个系统的适合我国国情的培养基质量控制国家标准,以致各相关单位采用的标准不一致,所以制定培养基质量控制国家标准非常关键。     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Dissociation and reconstitution of membranes synthesizing the peptidoglycan of Escherichia coli. Lipid dependence of the synthetic enzymes

The peptidoglycan synthetic enzymes can be dissociated with cholate and LiCl into components with mobilities on a gel filtration column in the same ranges as bovine serum albumin. The active enzymes can be separated further from the lipids necessary for synthesis by precipitation with ammonium sulfate. The needed lipids stable to hydrolysis with base. A protein needed for peptidoglycan polymerization can be separated from the other synthetic enzymes by hydroxylapatite chromatography.     

Detection and prevention of protein aggregation before,during, and after purification

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.     

Differentiation of DNA and RNA on filter paper discs

Differentiation of radioactive DNA and RNA deposited on filter paper discs can be accomplished by a relatively simple procedure. RNA can be efficiently removed by incubating the dises, impaled on pins, with 0.2 ml of 0.5 n NaOH for 90 min at 37°C. DNA can be removed after NaOH hydrolysis by treating the discs with 5% TCA for 30 min at 90°C. A correction is necessary to determine the actual amounts of DNA and RNA in order to account for the loss of DNA (13.8%) during the NaOH hydrolysis procedure.     

Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins

There are many factors that can influence the pharmacokinetics (PK) of a mAb or Fc-fusion molecule with the primary determinant being FcRn-mediated recycling. Through Fab or Fc engineering, IgG-FcRn interaction can be used to generate a variety of therapeutic antibodies with significantly enhanced half-life or ability to remove unwanted antigen from circulation. Glycosylation of a mAb or Fc-fusion protein can have a significant impact on the PK of these molecules. mAb charge can be important and variants with pI values of 1–2 unit difference are likely to impact PK with lower pI values being favorable for a longer half-life. Most mAbs display target mediated drug disposition (TMDD), which can have significant consequences on the study designs of preclinical and clinical studies. The PK of mAb can also be influenced by anti-drug antibody (ADA) response and off-target binding, which require careful consideration during the discovery stage. mAbs are primarily absorbed through the lymphatics via convection and can be conveniently administered by the subcutaneous (sc) route in large doses/volumes with co-formulation of hyaluronidase. The human PK of a mAb can be reasonably estimated using cynomolgus monkey data and allometric scaling methods.     

A protocol for isolation and culture of human umbilical vein endothelial cells

We describe a protocol for easy isolation and culture of human umbilical vein endothelial cells (HUVECs) to supply every researcher with a method that can be applied in cell biology laboratories with minimum equipment. Endothelial cells (ECs) are isolated from umbilical vein vascular wall by a collagenase treatment, then seeded on fibronectin-coated plates and cultured in a medium with Earles' salts and fetal calf serum (FCS), but without growth factor supplementation, for 7 days in a 37 degrees C-5% CO2 incubator. Cell confluency can be monitored by phase-contrast microscopy; ECs can be characterized using cell surface or intracellular markers and checked for contamination. Various protocols can be applied to HUVECs, from simple harvesting to a particular solubilization of proteins for proteomic analysis.     

A convenient and robust method for construction of combinatorial and random mutant libraries

Here we describe a convenient and robust ligase-independent method for construction of combinatorial and random mutant libraries. The homologous genes flanked by plasmid-derived DNA sequences are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of flanking primers, followed by assembly of the chimeric genes and linearized vector by PCR to introduce recombinant plasmids of a combinatorial library. Commonly, it is difficult to find proper restriction sites during the construction of recombinant plasmids after DNA shuffling with multiple homologous genes. However, this disadvantage can be overcome by using the ligase-independent method because the steps of DNA digestion and ligation can be avoided during library construction. Similarly, DNA sequences with random mutations introduced by error-prone PCR can be used to construct recombinant plasmids of a random mutant library with this method. Additionally, this method can meet the needs of large and comprehensive DNA library construction.     

Diploidy, evolution and sex

A new gene for a new purpose may be created by mutation of a pre-existing gene. But if that original gene is still required for its original purpose, and is to be retained side by side with the new, a spare copy is needed initially as raw material for the innovation. Thus in haploids the original gene must be duplicated before it is modified. But in diploids a spare copy of every gene is always available, and a mutant allele serving a new purpose can be easily established and maintained by heterosis in parallel with the old allele. Subsequent gene duplication will lead, via crossing-over, to insertion of the new gene in tandem with the old, as a permanent addition to the genome. Calculations show that diploids can thus enlarge their genomes with new genes for new purposes much more readily than haploids; in particular, they can more easily evolve the complex gene control systems characteristic of differentiated multicellular organisms. Sexual reproduction preserves diploidy, and so can be seen as the basis of these richer possibilities for evolutionary innovation.     

Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols

Problems inherent in the isolation of thiols from natural sources, such as oxidation, undesirable addition reactions, and low concentration of thiol species in cell-free extracts, can be circumvented by reversible derivatization to a less labile form which can be concentrated selectively. These objectives are realized by converting thiols to heterodisulfides in which the thiol partner is an apolar thiol with strong affinity for hydrophobic stationary phases. When reacted with 2-S-(2(')-thiopyridyl)-6-hydroxynaphthyldisulfide at pH<5, where most thiol species are relatively stable to atmospheric oxidation, mixed disulfides with 2-mercapto-6-hydroxynaphthalene as the apolar partner are obtained in good yield and can be concentrated onto a hydrophobic stationary phase. Such heterodisulfides exhibit excellent chromatographic properties when separated on reversed-phase media and the derivatization reaction can, therefore, be conveniently monitored. Following their isolation as the heterodisulfides the thiol species of interest are recovered by reduction and facile separation from the apolar 2-mercapto-6-hydroxynaphthalene partner.     

Simultaneous Separation and Purification of Neurofilament and Glial Filament Proteins from Brain

Neurofilaments (NF) and glial filaments (GF) were purified from bovine brain by the axonal flotation method, followed by hydroxylapatite chromatography in 8 M-urea. The proteins were shown to be competent to reassemble into intermediate filaments with removal of the denaturant, and reassembly was used as the final step in the purification of the filament proteins. The reassembly was found to be dependent on ionic strength and pH. This dependence was greater for neurofilaments than for the glial filaments. The NF and GF preparations were found not to be contaminated with each other by their gel electrophoretic profile and their immunological distinctness. The filament proteins can be obtained in high yield, and remain in solution if the urea is removed by dialysis against a low-ionic-strength buffer. Hence, they can provide a source for further biochemical studies.     

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320?nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light.     

Serum amyloid A and protein AA: Molecular mechanisms of a transmissible amyloidosis

Systemic AA-amyloidosis is a complication of chronic inflammatory diseases and the fibril protein AA derives from the acute phase reactant serum AA. AA-amyloidosis can be induced in mice by an inflammatory challenge. The lag phase before amyloid develops can be dramatically shortened by administration of a small amount of amyloid fibrils. Systemic AA-amyloidosis is transmissible in mice and may be so in humans. Since transmission can cross species barriers it is possible that AA-amyloidosis can be induced by amyloid in food, e.g. foie gras. In mice, development of AA-amyloidosis can also be accelerated by other components with amyloid-like properties. A new possible risk factor may appear with synthetically made fibrils from short peptides, constructed for tissue repair.     

Detection and manipulation of biomolecules by magnetic carriers

The detection and manipulation of single molecules on a common platform would be of great interest for basic research of biological or chemical systems. A promising approach is the application of magnetic carriers. The principles are demonstrated in this contribution. It is shown that paramagnetic beads can be detected by highly sensitive magnetoresistive sensors yielding a purely electronic signal. Different configurations are discussed. The capability of the sensors to detect even single markers is demonstrated by a model experiment. In addition, the paramagnetic beads can be used as carriers for biomolecules. They can be manipulated on-chip via currents running through specially designed line patterns. Thus, magnetic markers in combination with magnetoresistive sensors are a promising choice for future integrated lab-on-a-chip systems.     

Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags

Here, we present a cloning strategy for the production of recombinant proteins tagged with a polyhistidine sequence that can be cleaved by the exopeptidase, DAPase. The method can be used with most commonly available vectors and results in the expression of a His-tag protein that can be purified in its native form regardless of its natural sequence. This approach takes advantage of the TAGZyme system for the removal of amino-terminal affinity tags. Tag removal is accomplished either with DAPase (a recombinant dipeptidyl peptidase) alone or in combination with two accessory enzymes, Qcyclase and pGAPase. The system has been used for the production of intracellular proteins in Escherichia coli and can be applied to other expression hosts for the production of secreted proteins or proteins that require post-translational modification. The production of human interleukin 1beta in E. coli is used as an example to illustrate this method. The complete protocol from initial PCR to the production of a detagged protein with its authentic N terminus can be performed within 5 days.     

Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.     

Purification and kinetic characterisation of human erythrocyte actin

Human erythrocyte actin can be extracted from membrane ghosts by low ionic strength treatment in the presence of protective amounts of calcium and ATP. Purification then involves a single chromatographic step. The erythrocyte actin can be labelled with N-(1-prenyl)iodoacetamide. The fluorescence enhancement which accompanies polymerisation can be used to determine the critical concentration for assembly and to follow the polymerisation reaction time-course. The polymerisation kinetics of erythrocyte actin are compared with those of rabbit skeletal muscle actin. The two are shown to be markedly different.     

Applications of graph theory to enzyme kinetics and protein folding kinetics. Steady and non-steady-state systems

Graphic methods have proved to be very useful in enzyme kinetics, as reflected in both raising the efficiency of performing calculations and aiding in the analysis of catalytic mechanisms. The kinetic relations among protein folding states are very similar to those between enzyme-catalyzed species. Therefore, it should be equally useful to provide a visually intuitive relation between kinetic calculations and folding mechanisms for protein folding kinetics, as manifested by the graphic rules in enzyme kinetics. It can actually be anticipated that, due to increasing interest in protein folding, the graphic method will become an important tool in folding kinetics as well. Based on the recent progress made in graphic methods of enzyme kinetics, in this review four graphic rules are summarized, which can be used to deal with protein folding systems as well as enzyme-catalyzed systems. Rules 1-3 are established for deriving the kinetic equations for steady-state processes and Rule 4 for those in the case of non-steady-state processes. In comparison with conventional graphic methods, which can only be applied to a steady-state system, the current rules have the following advantages: (1) Complicated and tedious calculations can be greatly simplified. (2) A lot of wasted labor can be turned away. (3) Final results can be double-checked by a formula provided in each of the graphic rules. (4) Transient kinetic systems can also be treated. The mathematical proof of Rules 1-4 is given in appendices A-D, respectively.