摘要
Background To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.Methods Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from the duodenal mucosa of a bovine obtained at a wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His · Tag affinity chromatography, and its bioactivity was analyzed.Results Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DMA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using a Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 211 overnight and shown to have a high autocatalytic cleavage activity.Conclusion The EKL gene from a Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.
Background To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.Methods Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from the duodenal mucosa of a bovine obtained at a wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His · Tag affinity chromatography, and its bioactivity was analyzed.Results Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DMA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using a Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 211 overnight and shown to have a high autocatalytic cleavage activity.Conclusion The EKL gene from a Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.
基金
This study was supported by a National ' 95 ' Key Project grant (No. 96-C02-01-04).
