A method for developing a tissue proteome library
A technology of proteome and protein, applied in the field of obtaining tissue proteome library, can solve the problems of low success rate, unclear basis of technical and biological factors, etc.
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Embodiment 1
[0065] mRNAs for several proteins were collected from the NCBI database and translated in all 5'→3' orientation reading frames using the mRNA / DNA→Protein Translation tool available on the ExPASy Proteomics Server; data for 25 mRNAs, terminations found in Codons and calculated protein / peptide masses are summarized in Table 1.
[0066] We collected the mRNA sequences of 100 database reporter proteins (ranging in size from 10 to 100 kDa), translated them in all three 5'→3' orientation reading frames using the translation tool available on the ExPASy proteomics server, and we Only one frame was observed to yield the full length protein. Additionally, stop codons occurred at an average frequency of 16 to 24 amino acids in the remaining two misreading frames. This means that cloning and expression of cDNA in-frame with these erroneous expression vectors yields truncated peptides in the size range between 16-24 amino acids or between 1.84-2.76 kDa. This is completely consistent wit...
Embodiment 2
[0076] Taking cues from "Example 1" above, we performed interesting experiments; using the "Materials and methods" described in "Example 3" below, we synthesized total cDNA from purified snake oocyte mRNA , Cloning the recovered cDNA library into pT7T3D directional cloning vector. We transformed the plasmid library into salt-inducible GJ1158 bacteria by electroporation and expressed the total transcript library. About 33% of the cloned cDNAs will be in the correct reading frame of the expression vector, resulting in the authentic protein, while the rest of the cDNAs cloned in the wrong reading frame will produce truncated peptides. If bacteria were able to generate a total protein library from such a library after expression, we would expect that the isoelectric point (pi) and mass (kDa) range of the expressed protein should be similar to that of the bacterial protein. Analysis of the total proteins of the "uninduced" and "induced" libraries was performed on 12% 2D PAGE (FIGS...
Embodiment 3
[0090] Encouraged by the above finding that bacteria are capable of expressing protein libraries, we attempted and successfully used proteomics methods to resolve a group of rare and low-abundance proteins (DNA-binding proteins) from proteins expressed from snake oocyte cDNA libraries and then used Proteomic methods characterize them. We anticipate that this practice should form the evidence and basis for the successful development of "tissue proteome libraries". The DNA binding proteins expressed in the snake oocyte cDNA library constituted only a small fraction of the tissue proteins. Because these are low-abundance proteins, we definitely need a lot of tissue to express the protein. We grew 90 liters of bacterial culture in batches of 10 liters, which were induced for protein expression at the appropriate culture growth level (optical density). Bacteria are harvested, homogenized in an appropriate native buffer in the presence of protease inhibitors, and soluble proteins ...
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