Studies On Expression Of Recombinant Human ApoC Ⅱ In Pichia Pastoris

The gene of Human apolipoprotein C-Ⅱ(apoC-Ⅱ) located in the 19q13.2, is a 3.4kb fragment, which has four exons and three introns, mainly expressed in human liver and intestine. The mature peptide of ApoCⅡis a single strand polypeptide cpmposed of 79 amino acids. The preview polypeptide of ApoCⅡcontains 101 amino acids, after cleavaged from signal peptide, it transformed into mature ApoCⅡ.With two multiformity, ApoCⅡ's pI is 4.86 and 4.69, without Cysteine and Serine, and the molecular mass is 9110D.As a cofactor activates lipoprotein lipase(LPL), ApoCⅡis one of the most important subclasses of the ApoCs. ApoCⅡcould activates LPL from various sources, inhabiting the liver's absorption of the CM and VLDL, sustaining the stability of the lipoprotein(LP), engaging in the cleaning procedure of the lipoprotein grain in blood, and protecting the vascular endothelial cell. ApoCⅡhas an important biological function in the procedure of the lipoprotein catabolism in the condition of rich glycerol, and play an important role in preventing hypertriglyceridemia and atherosclerosis.Because of the important role of ApoCⅡin the lipid metabolism and its functions of preventing, diagnosing and curing the diseases in clinic, ApoCⅡhas became the focus of the research of the Apos. However, the ApoCⅡis isolated form human plasma which is quite limited in quantity, this problem would hamper the development of the further research and clinical application. So, it is of practical importance to discuses the producing procedure of recombinant protein with genetic engineering technology.In this study, we cloned the ApoCⅡcDNA sequence and use it as the template of the PCR. After adding two restriction enzyme sites to the ApoCⅡcDNA sequence, it was cloned to the modified pPICZαvector. We chose the Pichia pastoris which not only has the easy operating benefits of the prokaryotic system, but also own the ability of the processability of the eukaryontic system in order to express the recombinant ApoCⅡ. The pPICZαvector was modified as follows: elimilate the base pairs between the KpnⅠand XbaⅠ, XbaⅠand TGA, not including the sites of the KpnⅠ, XbaⅠ, XbaⅠand TGA.1. Cloning of ApoCⅡcDNA sequenceWe first obtained the cDNA sequence encoding ApoCⅡby RT-PCR, in which the template RNA derives from the human liver. Then the purified PCR production was cloned to pMD18-T vector. The results of sequencing showed that the recombinant cloning vector pMD18-T-ApoCⅡwas constructed correctly.2. Construction of recombinant ApoCⅡexpression system(1) Construction of ApoCⅡexpression vector pPICZα-ApoCⅡ With the recombinant plasmid of pMD18-T-ApoCⅡas template, the sequence encoding ApoCⅡwas obtained by PCR. The endonuclease sites of XhoI and XbaI were added into the two ends of interested sequence. Clone the sequence to pPICZαvector after being digested with those two endonucleases. The results of sequencing demonstrated that the construct was correct.(2) Transformation pPICZα-ApoCⅡinto Pichia pastorisWe transformed purified pPICZα-ApoCⅡvector into Pichia pastoris and screened on YPD plates containing 100μg/ml Zeocin. After 48 hours, dozens of colonies of transformants formed.3. Expression of recombinant ApoCⅡin Pichia pastorisSelected several clones from YPD/Zeocin plates, and induced them by 0.5% methanol in BMMY media. And we also analyzed the expression product by the methods of activity determination and SDS-PAGE. Results: (1)All the selected 20 clones showed that the expression system is effective to express recombinant ApoCⅡprotein. (2)Through being detected the time course expression of positive clone, it was found that there was a cumulative effect of production during the first 3 days and reach the peak at the end; In the next two days, the expression of recombinant protein was steady; Later the production of recombinant protein began reduce. (3)Analyzing the purified recombinant protein by SDS-PAGE, we found a protein of 10 kDa molecular weight. These results indicated that the functional recombinant ApoCⅡcould be induced in methylotropic yeast expression system and the secreting of recombinant protein had cumulative effect, which increased depending on time change. (4)When the positive clone was incubated in BMMY media with different pH varing from 2.2 to 6.0, the secretion of recombinant protein had a highest yield at around pH 5.2.The results indicated that the effective expression system of recombinant ApoCⅡhad been constructed. The variant Pichia pastoris can secret recombinant ApoCⅡ, which has a molecular weight of 10kDa.