Functional Expression, Promoter Cloning And Analysis Of Pig Liver Esterase 1

Pig liver esterase （PLE, EC 3.1.1.1） is a multigene family composed of several isoenzymes and belongs to the serine hydrolase. It has been shown that PLE may play important roles in the hydrolysis and metabolism of most endogenous and exogenous lipids （including prodrugs）. Changes of expression level and activity of PLE in pigs may influence efficacies and side-effects of relevant veterinary drugs. Unfortunately, studies on PLE were focused on the field of organic synthesis and rarely involved in the relations between hydrolysis of PLE and efficacies as well as side-effects of veterinary clinical drugs.PLE1 accounts for a large proportion in the family, and is one of the most important members. Functional expression of PLE1 can not only provide pure enzymes with the original activity for the study on hydrolysis characters of PLE 1 toward veterinary drugs, but also build technology platform for functional expressions of the other PLE isoenzymes. The cloning and analysis of PLE1 promoter can be helpful for uncovering the molecular mechanism of PLE 1 regulation, which can further benefit the predictions that what will happen to the expression levels of PLE by the effects of endogenous and exogenous compounds, and thus what will happen to the efficacies and side effects of veterinary clinical drugs, which will provides the essential theory basis for clinical medication and will benefit to illuminating transcriptional regulatory mechanisms of the other PLE isoenzymes. The main study results were as follows.First, the functional expression of PLE1 was carried out. The total mRNA of pig liver was extracted, primers were designed based on the 5’and 3’homologous sequences of PLE 1, and several PLE isoenzymes including PLE1 were obtained by RT-PCR and T-A cloning. After double digestion and recycling, PLE1 was linked into the vector of pET15b by T4 DNA ligase and the recombinant plasmid was named pET15b-PLE1. E.coli OrigamiTM 2（DE3） cells were transformed with pGro7 and then were treated by CaC12. The production cells were named Origami-pGro7. Origami-pGro7 cells were transformed with pET15b-PLE1 and a positive clone was named Origami-pGro7-PLE1 after identification. pGro7 was immediately expressed by the addition of L-arabinose, and the expression of PLE1 was started by the addition of IPTG when the strain entered the log phase. With the assistance of pGro7, PLE1 was highly expressed in the form of soluble protein, and showed enzymatic activity after detection.An 1153 bp 5’upstream promoter sequence of PLE1 gene was first cloned by nested PCR. And then it was analysed by the using of bioinformatics methods, it was found that there is no typical TATA box or CpG island existing in the region. And the potential transcription factor binding sites were predicted, including C/EBP, SP1, USF, SRY, etc. Meanwhile,7 motifs were found, including EGF₁, VWFC₁, ANAPHYLATOXIN₁, etc. 5’-RACE was performed to identify transcription initiation site of the PLE1 gene, sequence analysis of 5’-RACE products revealed that a partial nucleotide sequence at position-56 bp from ATG translational start site was discovered and was supposed to be the transcription initiation site. The highest level of transcriptional activity was in fragment -253/+65bp which we designated as the minimal core promoter of the PLE1 gene. It can be found that a SP1-binding site （a GC box） and a C/EBP-binding site located within the minimal promoter of the PLE1 gene were important for transactivation of the promoter. The mutation analysis results revealed that the transcriptional activity level of mutational constructs introducing of mutation into SP1 and C/EBP decreased by 55% and 22%, respectively, compared with the promoter activity of the intact -253/+ 65bp. Additional mutation in the C/EBP element to SP1,which abolished both SP1 and C/EBP element, caused a decrease of 46%. EMS As and supershift assays showed that SP1, SP3 could specifically recognize and bind to the GC box, and C/EBPa and C/EBPβ proteins could specifically recognize and bind to the C/EBP-binding site. From the results of transactivation analysis, it could be found that SP1 and C/EBPβ manifested as strong activators in the transactivation of PLE1 promoter, however, C/EBPa exhibited as strong depressor and SP3 seemed stay as transcriptional inactive molecules in the transactivation of the PLE1 promoter. And when C/EBPβ, SP1 and/or SP3 co-transfected with C/EBPa, they all showed negative cooperations and down-regulated the promoter activity greatly.