Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin.

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

A global benchmark study using affinity-based biosensors.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Antibody microarray analysis of inflammatory mediator release by human leukemia T-cells and human non small cell lung cancer cells.

Cytokines and chemokines are responsible for regulating inflammation and the immune response. Cytokine and chemokine release is typically measured by quantitative enzyme-linked immunosorbant assay (ELISA) or Western blot analysis. To expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based Thermo Scientific ExcelArray Antibody Sandwich Microarrays. Each slide consists of 16 subarrays (wells), each printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the "sandwich" ELISA, in which an analyte protein is sandwiched between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked Thermo Scientific DyLight 649 Dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. In our experiments, the Jurkat cell line was used as a model for human T-cell leukemia, and the A549 cell line was used as a model for human non-small cell lung cancer. To evoke a cytokine/chemokine response, cells were stimulated with tumor necrosis factor alpha (TNFalpha), phorbol-12-myristate-13-acetate (PMA, TPA), and phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angiogenesis, and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-alpha, and MIP-1alpha, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell-culture supernatants for the profiling of cellular inflammatory mediator release.

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology.

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators' instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1+/-1.6 x 10(6)M(-1)s(-1) and 6.7+/-2.5 x 10(-2)s(-1), respectively, which corresponded to an average equilibrium dissociation constant (K(D) of 2.6+/-1.4 x 10(-8)M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.

Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: implications for the release of vitamin K-dependent proteins.

Vitamin K-dependent (VKD) proteins are modified by the VKD carboxylase as they transit through the endoplasmic reticulum. In a reaction required for their activity, clusters of Glu's are converted to Gla's, and fully carboxylated VKD proteins are normally secreted. In mammalian cell lines expressing high levels of r-VKD proteins, however, under- and uncarboxylated VKD forms are observed. Overexpression of r-carboxylase does not improve carboxylation, but the lack of effect is not understood, and the intracellular events that occur during VKD protein carboxylation have not been investigated. We analyzed carboxylation in 293- and BHK cell lines expressing r-factor IX (fIX) and endogenous carboxylase or overexpressed r-carboxylase. The fIX secreted from the four cell lines was highly carboxylated, indicating fIX-carboxylase engagement during intracellular trafficking. The r-carboxylase was functional for carboxylation: overexpression resulted in a proportional increase in fIX-carboxylase complexes that yielded full fIX carboxylation. Interestingly, the carboxylated fIX product was not efficiently released from the carboxylase in r-fIX/r-carboxylase cells, resulting in decreased fIX secretion. r-Carboxylase overexpression changed the ratios of intracellular fIX to carboxylase, and we therefore developed an in vitro assay to test whether fIX levels affect release. FIX-carboxylase complexes were in vitro carboxylated with or without excess VKD substrate or propeptide. These analyses are the first to dissect the rates of release versus carboxylation and showed that release was much slower than carboxylation. In the absence of excess VKD substrate/propeptide, fIX in the fIX-carboxylase complex was fully carboxylated by 10 min, but 95% was still complexed with carboxylase after 30 min. The presence of excess VKD substrate/propeptide, however, led to a significant increase in VKD product release, possibly through a second propeptide binding site in the carboxylase. The intracellular analyses also showed that the fIX carboxylation rate was slow in vivo and was similar in r-fIX versus r-fIX/r-carboxylase cells, despite the large differences in carboxylase levels. The results suggest that the vitamin K cofactor may be limiting for carboxylation in the cell lines.

The influence of acetaldehyde and glycosaminoglycans upon factor Xa- and factor X-deficient plasma.

The comparative effects of glycosaminoglycans and acetaldehyde (AcH)--glycosaminoglycan (GAG) mixtures upon Factor Xa- (FXa) and Factor X-deficient plasma (FXDP) have been studied by activated partial thromboplastin time (APTT) studies. Heparin at 0.025, 0.030, 0.04, and 0.05 U statistically prolonged the APTT when pre-incubated with FXa at 37 degrees C for 3 min prior to addition to FXDP and subsequent addition of Ca2+. Upon addition of 0.25, 0.375, and 0.5 microg heparin-6000 (H6k) to FXa, significant increases in APTT were observed. Similarly, profound increases in APTT were observed when 0.5, 0.75, and 1.0 microg heparin-3000 (H3k) was added to FXa. The chondroitin sulfates (CSA, CSB, CSC) had far less impact upon APTT with the FXa-FXDP system. In examining the effects of AcH-GAG mixtures upon the clotting factor, it was observed that 44.3 and 443 mM AcH synergistically prolonged the APTT in a statistically significant manner regardless of the order of premixing the three components. Hence, AcH may play a role in prolonging APTT in alcoholics. It synergistically prolonged APTT in concert with GAGs and FXa at the AcH levels used in this study. The effect of the GAGs upon FXDP is far less than its effect upon FXa.