Method for distinguishing transport function of protein employing non-radioactivity

A protein and radioactive technology, applied in the field of non-radioactive discrimination of membrane protein transport function, can solve the problems of mitochondrial membrane potential increase, cell growth stagnation, ANT2 deficiency, etc., and achieve the effect of easy purchase, short operation time and low price

Inactive Publication Date: 2014-08-13
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ANT2 lacks this property
Inhibition of ANT2 expression leads to cell growth arrest and increased mitochondrial membrane potential

Method used

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  • Method for distinguishing transport function of protein employing non-radioactivity
  • Method for distinguishing transport function of protein employing non-radioactivity
  • Method for distinguishing transport function of protein employing non-radioactivity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Overexpression and purification of ANT4 protein

[0033] Transfect the pET-22b ANT4 (wild type) plasmid into E. coli C41 and amplify the cells to extract ANT4.

[0034] 1.1 Expression of ANT4 protein and bacterial amplification

[0035] Clone the ANT4 gene into pET-22b ( figure 2 ) (Novagen Company) vector, the plasmid carrying the ANT4 gene was transfected into Escherichia coli competent C41 (DE3) (Avidis Company) for expression.

[0036] Construction of recombinant plasmid: pET-22b carrying ANT4 gene ( figure 2 ) (Novagen) vector construction method reference literature: Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in Escherichia coli, Ashish K Sharma, Shubhashree Mahalik, Chaitali Ghosh, Anuradha B Singh and Krishna J Mukherjee, AMB Express 2011 , 1:33.

[0037] The constructed plasmid carrying the ANT4 gene was transfected into Escherichia coli competent C41 (DE3) (Avidis Company) for express...

Embodiment 2

[0066] Detect whether the wild-type uncoupling protein UCP2 has the function of transporting GTP and GDP

[0067] The expression, purification, and application of the wild-type UCP2 in lipoprotein recombinants are all the same as those of the wild-type ANT4. The transport function of UCP2 is studied by preparing lipoprotein recombinants with GDP as the substrate, and adding GTP on this basis. Finally, the respective contents of GDP and GTP after translocation were analyzed by HPLC to infer the translocation function of UCP2. The following is the analysis of the results after UCP2 transfer. From the result graph ( image 3 ) It can be seen that with the increase of the translocation reaction time, the content of GDP gradually decreases to a certain extent while the content of GTP increases correspondingly. Since this experiment operates at 6 time points, it can be seen that the translocation on UCP2 is mainly between 5s and 30s. After 30 s the transport reaction reaches sat...

Embodiment 3

[0069] The method for non-radioactive discrimination of membrane protein transport function, the steps are: dissolving natural soybean egg yolk lecithin in chloroform at a ratio of 100 mg / mL, drying the solution evenly with nitrogen gas, and dissolving the separated natural soybean egg yolk egg again with ultrapure water Phospholipids, and ultrasonically obtained liposomes of natural soybean yolk lecithin; Membrane protein: adenine nucleotide transferase 4 (ANT4) or uncoupling protein UCP2 was mixed with natural soybean yolk lecithin treated in the previous step, Add the substrate ATP and other components that stabilize the liposome structure and shake well to obtain the lipoprotein body reconstitution mixture; according to the proportion of the total volume, the specific composition is: detergent 10%wt TritonX-114: 8%, 100mg / Liposomes prepared by mL ultrasound: 14%, 10mg / mL cardiolipin: 8%, 0.3μg / μL membrane protein: 13%, 0.1mM substrate (ATP): 1%, 0.1M PIPES buffer: 9% , ul...

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Abstract

The method of non-radioactive discrimination of membrane protein transport function, recording the peak area displayed by ADP and ATP under different conditions, according to the peak area of ​​ADP and ATP to obtain their relative amount after the transport reaction, combined with different time points of transport reaction to obtain ADP / ATP If the ATP content tends to decrease and the relative ADP content tends to increase as the reaction time increases, it can be judged that the membrane protein has the function of transporting ADP / ATP. This method is superior to the isotope tracer method used to study the transport function of membrane proteins, mainly because of its safety, simplicity, and low cost.

Description

Technical field [0001] The invention involves the technical field of membrane protein functional discrimination, especially a method of non -radioactive discrimination function. Background technique [0002] The study of membrane protein structure and function, as the basis of studying drug targeted, is essential for the development of diseases.At present, the general method of studying membrane protein function is mainly structured functional research, and biological information technology is widely used as the core method of the core method to predict the structure and functions of protein.First of all, by searching the sequence information of the target protein in the database and comparing, including the same source sequence, topology structure, functional group, evolution, specific function related sequence analysis, etc.On the basis of predictive structure, the functional research of membrane protein also mainly relies on various modern biological physical instruments, incl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 李冬海张文江雪源张辰宇
Owner NANJING UNIV
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