Overproduction and purification of sucrose-phosphate synthase proteins in Escherichia coli for preparation of its antibody
1997
Sugiharto, B. | Jumadiyono, H. | Sumadi (Universitas Jember (Indonesia). Fakultas Pertanian)
Two cDNAs (pSoSPS1 and pSoSPS2) encoded for sucrose-phosphate synthase (SPS; EC 2.4.1.14) proteins have been isolated from sugar cane leaves. The cDNAs can be used for synthesis of their proteins in Escherichia coli cells. We have constructed the SPS-cDNAs into pQE vectors and followed by transformation of the constructs in E. coli cells (strain JM105) for production of SPS-proteins. However, due to the presence of significant homology between these cDNAs, we digested SPS-cDNAs around 1.2 kb from C-terminal and inserted the fragment into appropriate pQE vector to produce the SPS-proteins with different in their amino acid sequences. As it was expected approximately 47.8 kDa and 42.7 kDa of SPS recombinant proteins were expressed from the SoSOS1 and SoSPS2 fragments, respectively. Following purification of the protein using affinity column of Ni-NTA resin (Qiagen), about 400 micron (Myu)g of protein were injected subcutaneusly into New Zealand white rabbit. After 7 weeks injection, the antibodies against the protein were raised in rabbit antiserum. Upon Western Blot analysis the presence of antibodies have been confirmed. These results indicate that overexpression of SPS-cDNAs in E. coli provide tool to produce their proteins easier than purification of the proteins from the plants
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