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- Hammarström, Martin, et al.
(författare)
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Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.
- 2002
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 11:2, s. 313-321
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Tidskriftsartikel (refereegranskat)abstract
- A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.
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- Lavén, Martin, et al.
(författare)
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Radionuclide Imaging of Miniaturized Chemical Analysis Systems
- 2004
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 76:23, s. 7102-7108
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Tidskriftsartikel (refereegranskat)abstract
- We propose radionuclide imaging as a valuable tool for the study of molecular interactions in miniaturized systems for chemical analysis. Sensitive and quantitative imaging can be performed with compounds labeled with short-lived positron-emitting radionuclides, such as C-11 and Ga-68, within selected parts of the system. Radionuclide imaging is not restricted to transparent materials since the relatively energetic positrons can penetrate high optical density materials. Experimentally, a radiotracer is introduced into the object of study, which is subsequently placed on a phosphor storage plate. After exposure, the plate is scanned with a laser and a digital, quantitative image can be reconstituted. To demonstrate the concept, three types of microstructures suited for integration in chemical analysis systems were imaged with C-11- and Ga-68-labeled tracers. The influence of factors such as geometry of the object and type of radionuclide on resolution and sensitivity was investigated. The resolution ranged from 0.9 to 2.7 mm (fwhm). Measuring low amounts of radioactivity in the three structures, 2-20 Bq could be detected, which corresponded to 2.3-500 amol or 2.4-110 pM tracer. The imaging approach was applied to study analyte concentration and sample dilution effects on the performance of a capillary extraction column integrated in an automated LC-ESI-MS system. The utility of the technique was further illustrated by imaging of microchannels in a zeonor plastic compact disk and in a poly(dimethylsiloxane) material for the study of nonspecific peptide adsorption.
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- Lindström, Martin, et al.
(författare)
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Socioeconomic differences in smoking cessation : The role of social participation
- 2000
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Ingår i: Scandinavian Journal of Public Health. - : SAGE Publications. - 1403-4948 .- 1651-1905. ; 28:3, s. 200-208
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: The aim of this study was to investigate whether psychosocial resources explain socioeconomic differences in smoking cessation and its maintenance. Methods: A subpopulation of 11,837 individuals from the Malmö Diet and Cancer Study interviewed in 1992-94, age range 45-64 years, was investigated in this cross-sectional study. A multivariate logistic regression model was used to assess relative risks of having stopped smoking, adjusting for age, country of origin, previous/ current diseases, and marital status. Results: An odds ratio of 1.9 (1.4-2.5; 95% CI) for men and 2.0 (1.4-2.7; 95% CI) for women of having stopped smoking was found for higher non-manual employees when compared with unskilled manual workers. A decrease in these odds ratios was found when social participation was introduced into the model. The other three social network and social support variables were non-significant. Conclusion: High social participation is a predictor of maintenance of smoking cessation. It seems possible to interpret parts of the socioeconomic differences in smoking cessation and its maintenance as a consequence of differing social network resources and social capital between socioeconomic groups.
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- Merlo, Juan, et al.
(författare)
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Diastolic blood pressure and area of residence: multilevel versus ecological analysis of social inequity
- 2001
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Ingår i: Journal of Epidemiology and Community Health. - : BMJ. - 1470-2738 .- 0143-005X. ; 55:11, s. 791-798
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Tidskriftsartikel (refereegranskat)abstract
- STUDY OBJECTIVES---To study geographical differences in diastolic blood pressure and the influence of the social environment (census percentage of people with low educational achievement) on individual diastolic blood pressure level, after controlling for individual age and educational achievement. To compare the results of multilevel and ecological analyses. DESIGN---Cross sectional analysis performed by multilevel linear regression modelling, with women at the first level and urban areas at the second level, and by single level ecological regression using areas as the unit of analysis. SETTING---Malmö, Sweden (population 250 000). PARTICIPANTS---15 569 women aged 45 to 73, residing in 17 urban areas, who took part in the Malmö Diet and Cancer Study (1991-1996). MAIN RESULTS---In the "fixed effects" multilevel analysis, low educational achievement at both individual (beta =1.093, SE=0.167) and area levels (beta =2.966, SE=1.250) were independently associated with blood pressure, although in the "random effects" multilevel analysis almost none of the total variability in blood pressure across persons was attributable to areas (intraclass correlation=0.3%). The ecological analysis also found an association between the area educational variable and mean diastolic blood pressure (beta =4.058, SE=1.345). CONCLUSIONS---The small intraclass correlation found indicated very marginal geographical differences and almost no influence of the urban area on individual blood pressure. However, these slight differences were enough to detect an effect of the social environment on blood pressure. The ecological study overestimated the associations found in the "fixed" effects multilevel analysis, and neither distinguished individual from area levels nor provided information on the intraclass correlation. Ecological analyses are inadequate to evaluate geographical differences in health.
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- Woestenenk, Esmeralda A., et al.
(författare)
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His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors.
- 2004
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Ingår i: Journal of structural and functional genomics. - 1345-711X .- 1570-0267. ; 5:3, s. 217-29
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Tidskriftsartikel (refereegranskat)abstract
- We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.
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- Woestenenk, Esmeeralda A., et al.
(författare)
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Screening methods to determine biophysical properties of proteins in structural genomics
- 2003
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Ingår i: Analytical biochemistry. - 0003-2697. ; 318:1, s. 71-79
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Tidskriftsartikel (refereegranskat)abstract
- We have developed and tested a simple and efficient protein purification method for biophysical screening of proteins and protein fragments by nuclear magnetic resonance (NMR) and optical methods, such as circular dichroism spectroscopy. The method constitutes an extension of previously described protocols for gene expression and protein solubility screening [M. Hammarström et al., (2002), Protein Science 11, 313]. Using the present purification scheme it is possible to take several target proteins, produced as fusion proteins, from cell pellet to NMR spectrum and obtain a judgment on the suitability for further structural or biophysical studies in less than 1 day. The method is independent of individual protein properties as long as the target protein can be produced in soluble form with a fusion partner. Identical procedures for cell culturing, lysis, affinity chromatography, protease cleavage, and NMR sample preparation then initially require only optimization for different fusion partner and protease combinations. The purification method can be automated, scaled up or down, and extended to a traditional purification scheme. We have tested the method on several small human proteins produced in Escherichia coli and find that the method allows for detection of structured proteins and unfolded or molten globule-like proteins.
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