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| High Level Prokaryotic Expression of Thymosin α1 by Gene Repeats |
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Abstract A new thymosin α1(Tα1) gene [SD-His.tag-EK- Tα1] named SD-Tα1 was synthesized, consecutively consisting of SD sequence, sequence encoding His.tag, enterokinase recognition site and Tα1 cDNA. This gene monomer was multiplied up to 8-tandems using Isocaudamer XbaI and SpeI. These different repeats of SD-Tα1 were inserted into plasmind pET-32a(+) respectively and then transformed into E. coli BLR(DE3) to achieve a series of genetic bacteria E. coli BLR(DE3) containing 1~8 copies of SD-Tα1.PCR analysis and the sequencing showed that 1~8 copies of SD-Tα1 were all correctly cloned into pET-32a(+) vector. Furthermore, the reporter gene lacZ with its own SD sequence (SD-lacZ) was added as a tail of SD-Tα1 tandems to verify the translation efficiency of the fusion gene through blue-white clone screening. The expected blue clones were obtained such as genetic E. coli BLR(DE3)strain containing pET32a-1Tα1-LacZ、pET32a-3Tα1-LacZ、pET32a-5Tα1-LacZ and pET32a-8Tα1-LacZ. The results implied that each SD-Tα1 repeat can be authentically transcribed and submitted for translation. Fermentation and SDS-PAGE analysis indicated that Tα1 peptide was expressed in a soluble form in all the genetic bacterial strains containing 1~8 repeats of SD-Tα1. In conclusion, a series of multiple repeats of Tα1 gene has successfully been constructed and expressed in E. coli with a new construction method. Also, a platform of high prokaryotic expression of small peptides was established.
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Received: 31 July 2006
Published: 10 May 2010
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