A quality comparison of protein crystals grown under containerless conditions generated by diamagnetic levitation, silicone oil and agarose gel.

High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

Effect of phenyl sepharose ligand density on protein monomer/aggregate purification and separation using hydrophobic interaction chromatography.

In the large-scale manufacturing and purification of protein therapeutics, multiple chromatography adsorbent lots are often required due to limited absorbent batch sizes or during replacement at the end of the useful column lifetime. Variability in the adsorbent performance from lot to lot should be minimal in order to ensure that consistent product purity and product quality attributes are achieved when a different lot or lot mixture is implemented in the process. Vendors of chromatographic adsorbents will often provide release specifications, which may possess a narrow range of acceptable values. Despite relatively narrow release specifications, the performance of the adsorbent in a given purification process could still vary from lot to lot. In this case, an alternative use test (one that properly captures the lot to lot variability) may be required to determine an acceptable range of variability for a specific process. In this work, we describe the separation of therapeutic protein monomer and aggregate species using hydrophobic interaction chromatography, which is potentially sensitive to adsorbent lot variability. An alternative use test is formulated, which can be used to rapidly screen different adsorbent lots prior to implementation in a large-scale manufacturing process. In addition, the underlying mechanism responsible for the adsorbent lot variability, which was based upon differences in protein adsorption characteristics, was also investigated using both experimental and modeling approaches.

The defined antigen substrate spheres (DASS) system and some of its applications.

A quantitative immunofluorescent technique based on the covalent coupling of protein to Sepharose beads, the so-called defined antigen substrate spheres (DASS) system is described, and the main technical aspects are discussed. The method proved to be reproducible and has a lower detection limit equivalent to the amount of antigen, present per bead, that can bind 1 pg fluorescent labeled antibody. The DASS system has many applications, and some of them are discussed, such as the quantitation of antibodies and antigens with Sepharose-coupled antigens or antibodies. Not only antigen-antibody interactions but also any reaction that results in a stable complex can be studied, provided antisera against one of the constituents are available. An example is given by the complement fixation to Sepharose-coupled IgG. For the quantitation of hydrophobic antigens, hydrophobic Sepharise can be used.

Microfluorometric evaluation of conjugate-specificity with the defined antigen substrate spheres (DASS) system.

Six fluorescent antihuman Ig preparations were tested for their Ig class specificity by reacting them with highly purified IgG, IgM, IgA, and OVA coupled covalently to Sepharose beads. OVA was used as a measure for nonimmunologic binding. Bead fluorescence was determined by microfluorometry. The amounts of USS and NSS were expressed quantitatively. These data were compared with the performance of these particular conjugates in a biologic system, namely, monoclonal bone marrow cells. Five of the six conjugates satisfied the requirement of monospecific activity; one did not. At a dilution of 1 : 8, the five monospecific conjugates reacted between five and 50 times stronger with their appropriate antigens than with OVA-coupled beads. Cross reactivity with other Ig classes, after correction for OVA staining was maximally 6%. The conjugate that was nonspecific in the bone marrow system gave very high cross reactivity with the Ig-coupled beads. A good correlation was found between OVA bead staining and nonimmunologic binding of conjugates in bone marrow slides. In this respect, conjugates prepared from antibody preparations isolated by solid immunoadsorbents proved to be superior to globulin or whole IgG fractions. Ig coupled to Sepharose beads seems to represent a very promising substrate for conjugate specificity testing.

Comparison of properties of agarose for electrophoresis of DNA.

Agarose as a medium for separation of DNA was first introduced in 1962 and since the early 1970s agarose submarine gel electrophoresis has been synonymous with separations of DNA molecules larger than 1 kilobase pair (kb). The large pore size, low electroendosmosis and strength of the matrix have advantages over other media such as polyacrylamide for many applications. The variety of grades of agarose, developed by chemical manipulation of the substitutions on the agarose polymer, provides a range of matrices for separation of DNA molecules from a few base pairs (bp) to over 5 megabase pairs (Mb) in length. The introduction of low-melting-temperature agarose has revolutionised the extraction and manipulation of chromosome-sized molecules. On the other hand, the demand for analysis of very small quantities of DNA will most likely lead to the increasing importance of capillary electrophoresis. Many theories have been propounded to explain the electrophoretic migration of DNA in agarose. The most popular of these has been reptation theory but none can account for all of the reported anomalies in migration. However, anomalous migration has been exploited to study DNA structure, topology and catenation. An example of the use of two-dimensional electrophoresis to demonstrate the complexity of DNA migration through agarose is given. Generally, for molecules smaller than 50 kb, electrophoretic separation is a function of length. By alternately electrophoresing DNA in two different directions, molecules as large as 5.7 Mb have been effectively separated, although with such large molecules DNA structure as well as size may determine migration. In the case of separations of chromosomes from the intestinal protozoan, Giardia duodenalis, for example, a discrepancy of 1 Mb in the size of one chromosome, with an apparent size of 0.7-2.0 Mb, depended on the boundary conditions of separation. Major challenges for the molecular biologist are separation of larger chromosomal sized molecules, greater number of samples and smaller formats. Towards this challenge computer-aided technology is a key component in the control of electrophoresis parameters and analysis.