PICln Gene Cloning And Construction Of Pichia Pastoris Intracellular Expression Vector

Subject:pICln gene cloning and construction of pichia pastoris intracellular expression vectorAuthor:He LiMajor:Biochemistry and Molecular BiologyTutor:Yingzhe Ma ProfessorVolume regulation is the basic physiological function of cell, In the condition of swelling or shrinkage for long time, Cell even can apoptosis by dysfunction.When cells swollen under the influence of some factors, cell itself can regulate their volume back to normal through a variety of regulatory mechanisms.One of the regulatory way is Chloride channel which controls the inside ions and organic substances out of the membrane by open or off state of channel protein in plasma membrane. At present, there are four candidate proteins of Chloride channel protein, namely P-glycoprotein, ClC-2, ClC-3, and pIcln which the object of this paper.pICln is a ubiquitous and abundant 27-43KDa soluble protein, and over-expression can induce an outwardly rectifying anion current. It is an acidic protein that contains three acidic amino acid region. P107-152 peptide, an 46 amino acid sequence present in the second acidic region. The expression of only P107-152 was enough for the cells to acquire the maximal 'survival rate'. The hypotonic resistance enhanced by expression of this peptide was as strong as that of the full-length pICln.The increased viability by expression of P107-152 was inhibited by extra-cellular ATP and anion channel inhibitor DIDS.So this peptide is the functional areas to regulate cell volume and resist hypotonic environment.The functionality research on pICln is mainly in two aspects. On the one hand, it can transport the intracelluar ion to resist the hypotonic environment and maintain normal volume of cell. pICln "channel" was a homodimer, and that each monomer contained four P strands that contribute to the formation of an eight-stranded, antiparallelβbarrel transmembrane pore. Then, other points of mechanisms on cell volume adjustment function appeared.In 1994, Krapvinsky et al. found pICln mainly localized in the cytoplasm, not in the plasma membrane, indicating that it is not channel protein, but overexpression of pICln can induce an apparently outwardly Rectifying Chloride Current, considering it is chloride channel regulator presented in the cytoplasm. Later, the third mechanism hypothesis was proposed because of the founding that pICln can naturally bind and separated with actin which is the component of cytoskeletal. Binding of actin,pICln plays a nature ion channel function when hypotonic stimulate cell swelling.while cell volume returne to normal state, pICln separate with actin and then back to the cytoplasm. pICln as a chaperone protein in involved in mRNA splicing process for its second effect. pICln combine with PRMT5 which has a variety of illicit substrates in the cytoplasm, enhancing the affinity between PRMT5 and the spliceosome Sm to the spliceosome components of Sm protein, then PRMT5 responsible for generating symmetric dimethylarginine modifications on the RG-rich region of SmDl, SmD2, SmB. Subsequently produced a mature snRNPs,then snRNPs come into the nucleus and combine to hnRNA for RNA splicing process.No matter what kind of mechanism to play the role of chloride channel, the function of maintaining cell volume is very important. pICln protect cell to resist hypotonic in E. coli cells,then we pay our attention on organelles more completed eukaryotic yeast cells.So,we choose pichia pastoris cells as the exogenous expression host of pICln. Cloning pICln gene and construction of pichia pastoris intracellular expression vector, then transfected into yeast cell.It is the foundation of research on pICln that have the function of anti-swelling in eukaryotic yeast cell.pICln was expressed in many kinds of tissues and cells, and it had a high homologous in different species. And renal have more expression of pICln. This is the reason that we choose renal tissue as our material for extracting total RNA. reverse transcription of total RNA extraction and PCR to gain more cDNA coding pICln. We get the pICln CDS sequence of rat in Genebank,then design a pair of PCR primers by Primer 5.0, Selecting two small DNA fragment Outside the range of initiation codon and termination codon. In the 5'end of upstream primer added BamHI restriction enzyme sites and downstream primer added EcoRI restriction enzyme site. PCR products were used to construction the clone vector after purified. Positive colonies were selected by blue/white screening assay. Culture the colony and identify the vector in it by PCR and sequencing. After these steps the cloning vector was successfully constructed, and named pTG19-T-pICln. Double digestion the intracellular expression vector and the target fragment. Then ligated them after recycling. Positive clones were screened using ampicillin and identified by PCR, digestion and sequencing. The Yeast cell expression vector was constructed, named pPIC3.5K-pICln. Transfected Pichia pastoris SMD1168 and made the target fragment expressed in it. After that the pICln gene can be studied about the role of anti-swelling in eukaryotic cell.