BACKGROUNDTo 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.

METHODSBovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from duodenal mucosa of a bovine obtained at 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 it bioactivity was analyzed.

RESULTSCompared 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 DNA 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 Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21 degrees C overnight and shown to have a high autocatalytic cleavage activity.

CONCLUSIONSThe EKL gene from 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.

Animals ; Catalytic Domain ; genetics ; Cattle ; Cloning, Organism ; DNA, Complementary ; Enteropeptidase ; analysis ; genetics ; Recombinant Proteins

AIMTo detect the residual clenbuterol in pork meat and liver using HPLC with Coulometric electrode array system.

METHODSHomogenized meat or liver sample was treated with 1 mol x L(-1) hydrochloric acid and centrifuged, the fat existing in meat or liver tissue was removed by diethyl ether. The pH of the remaining aqueous layer was adjusted to 10.8 +/- 0.2 or 11.6 +/- 0.2 for meat or liver and liquid-liquid extraction with diethyl ether was followed. The ether extract was evaporated to dryness, the residue was dissolved in the mobile phase. The mobile phase A consisted of 50 mmol x L(-1) phosphoric acid-30 mmol x L(-1) triethylamine and was adjusted to pH 4.0 with 2 mol x L(-1) sodium hydroxide solution. The mobile phase B consisted of methanol-acetonitrile (30:45). A mixture of mobile phase A and B (80:20) was used in the method. A four electrode array module was selected for quantitation, the electrode potentials were set at 450, 600, 650 and 680 mV respectively.

RESULTSThe two calibration curves for meat and liver showed good linearity between 1.88 - 60.16 ng x g(-1), the detection limit of clenbuterol was 1.2 ng x g(-1).

CONCLUSIONThis method using HPLC-electrochemical detection is reproducible, and the sensitivity is good enough for the determination of clenbuterol in meat and liver.

Animal Feed ; analysis ; Animals ; Chromatography, High Pressure Liquid ; methods ; Clenbuterol ; analysis ; Drug Residues ; analysis ; Electrochemistry ; methods ; Electrodes ; Liver ; chemistry ; Meat ; analysis ; Swine

Air Pollution ; Asthma/drug therapy* ; China ; Humans ; Treatment Adherence and Compliance