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Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins.
We have modified this technique for the isolation of plant calmodulin-binding proteins. [35S]-methionine was used instead of the inorganic [35S]-sulfate, or125I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure
thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large
number of filters. Here we describe in detail a procedure for the production and purification of [35S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The
[35S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II
that was previously isolated using a [125I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive
clone encoding a calcium-dependent calmodulin-binding protein was isolated. 相似文献
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Recombinant human interleukin 1 alpha: purification and biological characterization 总被引:29,自引:0,他引:29
U Gubler A O Chua A S Stern C P Hellmann M P Vitek T M DeChiara W R Benjamin K J Collier M Dukovich P C Familletti 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(7):2492-2497
Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1. By using the murine IL 1 cDNA as a probe, we have isolated its human homolog from cDNA generated to lipopolysaccharide-stimulated human leukocyte mRNA. Nucleotide sequence analysis of this cDNA predicts a protein of analysis of this cDNA predicts a protein of 271 amino acids (termed IL 1 alpha) which shows congruent to 61% homology to its murine counterpart but only 27% homology to a recently characterized human IL 1 precursor (IL 1 beta). We have expressed the carboxy-terminal 154 amino acids of IL 1 alpha in E. coli, purified this protein to homogeneity, and have compared it with pure recombinant murine IL 1 in several different IL 1 assays based on murine and human cells. Recombinant IL 1 is capable of stimulating T cell and fibroblast proliferation and inducing fibroblast collagenase and prostaglandin production, thus proving that a single molecule has many of the activities previously ascribed to only partially purified IL 1 preparations. Our results indicate that there exists a family of at least two human IL 1 genes (alpha and beta) whose dissimilar protein products have similar biological activities. 相似文献
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Purification and preservation of potato virus X from leaf sap of tobacco plants before lyophilization was carried out by two methods: 1) precipitation by polyethylene glycol and ultracentifugation, and 2) precipitation by ammonium sulphate, chromatography on Sephadex G-50 and ultracentrifugation. The first method is preferable to the second because the final preparation contains more virus antigen. Both preparations were strongly infectious and maintained antigenic properties after lyophilization. To achieve a more gentle course of lyophilization of virus preparations, addition of urotropine and dextran T-10 to the virus suspension, purified by the precipitation by polyethylene glycol-6000, was examined. Addition of urotropine was proved unsatisfactory, because only antigenic properties were maintained after lyophilization while the infectivity disappeared. But we can recommend addition of dextran T-10 up to a concentration of 6% to the preparation of virus antigen before lyophilization. The course of lyophilization is much rapider, the lyophilized product can be very easily dissolved in water and is not hygroscopic. The product is strongly infectious and gives the serological precipitation reaction in a dilution four times that of X virus antigen lyophilized without addition of dextran T-10. 相似文献
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