Nikonorova A strategy for the selection of monovalent antibodies that span protein dimer interfaces Author Profile: Jamie B. Spangler Author Profile: Ignacio Moraga Editors' Picks Highlight2 Catching a complex for optimal signaling JBC Reviews Mitochondrial plasticity in cell fate regulation Letters to the Editor Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact Reply to Yamauchi et al. Sign up for eTOCs Electronic table of contents alerts are a free service providing table of contents or a notification of availability when new issues of The Journal of Biological Chemistry are published online.
Sign Up. Submit your work to JBC. The vector with or without fusion partners was digested with Ssp1 for two hours and applied to DNA agarose electrophoresis. Positive clones were screened by PCR and then sequenced.
The E. A single clone was picked and inoculated into 3 mL LB media for overnight growth. The lysate was fractionated by centrifugation for 20 min at 10, g. The supernatant normally contained soluble proteins and fragmented membranes, while the pellet consisted of insoluble proteins inclusion body fraction. Control experiments were performed under the same experimental conditions without IPTG induction. MBP-Rvc was expressed in 10 mL cultures as described above. These proteins range in molecular weight from 7. This substantial increase in cloning efficiency can be attributed to the simplicity of ligation independent cloning.
The most difficult step in the ligase dependent method is ligation, whose efficiency is strongly influenced by the sequence of the sticky end, the efficiency of the restriction endonuclease enzyme digestion and the experimental conditions. For LIC the ligation is performed in vivo by the intracellular system in the host cell, thereby avoiding the in vitro difficulties and increasing the cloning efficiency.
LIC has been successfully used in high throughput cloning for soluble proteins [ 16 ], and the cloning efficiency in the present work is close to that for soluble proteins, indicating that this LIC method is also effective for high throughput cloning of membrane proteins. Table 1 summarizes the results of the first round expression screening.
Of the 39 cloned genes, 30 of them could be expressed as detected by Coomassie blue staining or western blot. Overexpression of 16 putative membrane proteins in the first round of expression screening. The gels were stained by Coomassie blue and the expressions were confirmed by western blot using an antibody against the His-tag.
The arrows indicate the expressed proteins for lanes where it is not obvious. As in the previous study [ 2 ], it was found that many overexpressed membrane proteins were in the inclusion body fraction. The advantage of such expression is that the protein is often produced in large quantity sometimes half of the whole cell protein mass and in relatively pure form. Proteins expressed in inclusion bodies are also protected from proteolysis. Despite these advantages there are concerns about the refolding of proteins from inclusion bodies.
The work from Villaverde's group showed two important features of these precipitated aggregates, namely that they are structurally and dynamically heterogeneous, but importantly, they provided evidence that the aggregation in the inclusion bodies is reversible [ 25 ], consistent with the recent refolding efforts. Expression in the membrane fraction is a strong indication that the protein may be in its native or native-like conformation and that the in vitro refolding process can be avoided in sample preparations for various characterizations.
To improve upon the first round efforts, three commonly used fusion partners MBP [ 26 ], GST [ 27 ] and KSI [ 28 ] were tested for the 10 putative membrane proteins that did not express in the first round of screening.
Table 2 lists the results for cloning and expression screening of the fusion proteins where expression was detected by Coomassie blue staining. Since the same LIC site was used in all the vectors Figure 1 , T4 DNA polymerase treated PCR products in the first round of screening were inserted directly into these vectors without any additional treatment.
All the target genes were successfully inserted into the fusion protein vectors and expressed at high level from at least one of the fusion vectors. Figure 3 shows the gels for expression of Rv c and Rv from the various vectors. Expression of Rv A and Rvc B in different constructs.
Cells were harvested and then sonicated W: whole cell. After centrifugation at 10, g for 20 min, the inclusion body fraction IB was collected. The supernatant was ultracentrifuged at , g for 45 min, and the supernatant soluble fraction S and pellet membrane fraction M were collected, respectively. The arrows indicate the overexpressed fusion proteins.
Note that Rv and Rvc could not be overexpressed without fusion partners, and there was no detectable expression for the KSI-Rv fusion. As expected, all of the KSI fusion proteins aggregated in the inclusion body fraction Table 2 , Figure 3. Of the ten tested membrane proteins, Rv was the only one that was exclusively expressed as a KSI fusion protein. Kcv protein, a viral potassium channel, is another example where KSI fusion has been uniquely successful Qin and Gao, unpublished data.
GST is a highly soluble protein and frequently used as a fusion partner to increase the solubility and the yield of small soluble proteins. GST has been used to fuse with a low molecular weight membrane protein, PsbH [ 30 ], and it was found that the majority of the fusion protein was in a soluble state, facilitating the purification by affinity chromatography with immobilized glutathione resin. However, in our experiments, although the molecular weights of the target proteins were less than 16 kDa, all of the expressed fusion proteins were obtained in the inclusion body fraction Table 2 and Figure 3.
Methods in Membrane Biology - Volume 1 | Edward D. Korn | Springer
It has been previously reported that the capability of GST to increase the solubility of the passenger proteins was weak among the commonly used fusion partners [ 31 , 32 ] consistent with our results. Compared with KSI fusion proteins, GST fusion proteins could be relatively easily solubilized and refolded in detergent micelles, facilitating the cleavage by a specific protease to release the passenger protein prior to further purification [ 10 , 22 ].
MBP is known for its strong capability to increase the solubility of the target protein and has been extensively used as a fusion partner for both soluble and membrane proteins [ 7 , 8 , 26 , 27 ]. It is not surprising that MBP increases the solubility of the target protein, but how the MBP fusion tag enhances affinity for the membrane fraction is unclear.
Even when the high molecular weight of MBP is taken into account, the expression of the target membrane proteins are still substantially higher than those for KSI and GST fusion proteins. Both features make MBP an attractive fusion partner for membrane proteins: the target membrane proteins are not only overexpressed, but also in the native membrane.
Based on these and previous results [ 7 , 8 ], the application of MBP as a fusion partner for membrane proteins is a powerful tool for the study of membrane proteins. Korn at Barnes Noble. Books Division. Chicago Distribution Center. Examination of the tables of contents of journals - biochemical, molecular biological, ultrastructural, and physiological-provides convincing evidence that membrane biology will be in the s what biochemical genetics was in the s.
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Materials and Methods Edited by Edward D. Development of a method of isolating single vertebrate nerve fibers and of measuring electrical properties of the myelin sheath and node of Ranvier. Methods in Membrane Biology, Vol. Walter H, Krob EJ. Partition of cells defined as the percentage of added cells in the top phase in dextran-polyethylene glycol aqueous two-phase systems is an extremely sensitive indicator Edward d.
MARY G. From the Section on A method is described for the rapid and efficient isolation of phagocytic vesicles from large. Korn: Books. Edward d. Introduction The translocation of proteins into and across biological membranes requires the coordinated action of multiple components for a review, see articles in Neupert and Lill, Although not the only volume in this series in which lipids are discussed, the present volume is devoted entirely to methods for the study of membrane lipids.
Even now, when membrane proteins are properly receiving so much attention, this emphasis on membrane lipids is appropriate.
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