ALBERT

Description: Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 517-526, 2002.

Description: With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

Description: The architecture of NASA's program of robotic Mars exploration missions received an intense scrutiny during the summer months of 1998. We present here the results of that scrutiny, and describe a list of Mars exploration missions which are now being proposed by the nation's space agency. The heart of the new program architecture consists of missions which will return samples of Martian rocks and soil back to Earth for analysis. A primary scientific goal for these missions is to understand Mars as a possible abode of past or present life. The current level of sophistication for detecting markers of biological processes and fossil or extant life forms is much higher in Earth-based laboratories than possible with remotely deployed instrumentation, and will remain so for at least the next decade. Hence, bringing Martian samples back to Earth is considered the best way to search for the desired evidence. A Mars sample return mission takes approximately three years to complete. Transit from Earth to Mars requires almost a single year. After a lapse of time of almost a year at Mars, during which orbital and surface operations can take place, and the correct return launch energy constraints are met, a Mars-to-Earth return flight can be initiated. This return leg also takes approximately one year. Opportunities to launch these 3-year sample return missions occur about every 2 years. The figure depicts schedules for flights to and from Mars for Earth launches in 2003, 2005, 2007 and 2009. Transits for less than 180 deg flight angle, measured from the sun, and more than 180 deg are both shown.

Description: NASA's Mars Surveyor Program (MSP) will launch two mission to the red planet about every 26 months (determined by energy considerations) from 1996 through 2005 at an annual cost of $100 million dollars per year (excluding the launch vehicles). Mars Global Survey (1996) and Mars Surveyor 98 are described in other papers. This paper will focus on the planning that is under way for the MSP missions to be launched in 2001 and beyond.

Description: Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Description: The objective of this research is to determine the effects of thermal processing, freeze drying, irradiation, and storage time on the nutritional content of food, to evaluate the nutritional content of the food items currently used on the International Space Station and Shuttle, and to establish the need to institute countermeasures. (This study does not seek to address the effect of processing on nutrients in detail, but rather aims to place in context the overall nutritional status at the time of consumption).

Description: This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the analyte were in the sub-microMolar range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins. c2003 Elsevier B.V. All rights reserved.

Description: In the not too distant future, NASA may consider sending a robotic mission to Mars to drill tens of meters below the surface to search for evidence of life. Mars science groups, including NASA's Mars Exploration Program Analysis Group (MEPAG), have repeatedly concluded that in situ scientific analyses of samples from significant depths below the surface are important for understanding Mars in general and for searching for evidence of past or present life in particular. Furthermore, there are several ongoing technology developments for relevant drills, the readiness of which seem promising for use by the second decade of this century. By accessing and analyzing material from tens of meters below the surface, in situ science investigations may help answer some important questions about Mars, in particular about whether life ever existed there. Drilling is a proven technique for terrestrial applications that appears viable for accessing Martian subsurface samples and bringing them to the surface for analysis by a variety of instruments. An end-to-end mission concept for a Deep Drill mission has been developed and appears feasible for launch in the next decade.