Cloning And Expression Of HSLPI In Pichia Pastoris

SLPI(secretory leukocyte peptidase inhibitor)is one of the two members ofthe ALP superfamily of proteinase inhibitors and a kind of whey acidic protein(WAP) motif protein. The mature SLPI consists of 107 amino acids, which iscleavaged from 14.3kDa SLPI precursor with a signal peptide of 25 amino acids.SLPI is a non-glycosylated, hydrophobic, highly basic (pI >. 9.5), acid-stable,cationic 11.7-kDa single-chain polypeptide serine protease inhibitor, relativelyheat-stable, consisting of two homologous cystein-rich domains of 53 and 54amino acids. The human SLPI gene is localized on chromosome 20q12-13.2.The SLPI gene consists of four exons and three introns and spans approximately2.6 kb, which can be modulated at both the transcriptional and translationallevels. To date, no polymorphism of the SLPI gene and no state of SLPIdeficiency have been found. The SLPI gene belongs to the trappin gene family.The products of this family are characterized by an N-terminal transglutaminasedomain substrate and a C-terminal four-disulfide core. These two domains(COOH terminal and NH2 terminal) share about 35% homology. Each of thesedomains has distinct enzyme activities. The tertiary structure of the SLPImolecule resembles a boomerang, with each arm carrying one domain. Thefour-in-each-domain disulfide bridges formed between the 16 cysteine residues,as well as the two-domain interaction, thereby connecting the polypeptidesegments of the molecule, contribute to the conformation and efficacy of themolecule.SLPI is a potent inhibitor of a variety of endogenous proteolytic enzymes andcould be in host defence against invading micro-organisms, and even the DNAduplication of virus. Along with the more and more understanding of thisprotein, people have realized that it is an innate immune reaction for SLPI toprotect local mucus membrane and tissue against the inflammatory injuries. It isgenerally postulated that the balance between proteinases and antiproteinases isa prerequisite for the maintenance of tissue integrity. Indeed, it is showed thatcleavage of SLPI results in increased tissue damage. The protease inhibitoryactivity is restricted to the COOH-terminal domain with the active center beingformed by the [Leu.sup.72]-[Met.sup.73] residues. The NH2-terminal domain(N-terminal domain) has no such properties, but it may aid in stabilizing theprotease-antiprotease complex and may mediate the enhancement of theantiproteinase activity of SLPI by heparin. Heparin augments the effectivenessof SLPI as it induces a conformational change in the inhibitor. It seems that theN-terminal domain is responsible for the dose-dependent bactericidal propertiesof SLPI against both gram-positive (S. aureus) and gram-negative (E. coli)bacteria.Recent scientific evidence suggests that SLPI has broad-spectrumantibiotic activity that includes bactericidal and antifungal properties. As withthe antibacterial-bactericidal activity, the antifungal activity was mainlylocalized in the NH2-terminal domain. It is believed that killing of fungusprotects the epithelia from the fungal proteases. Probably the antibacterial andantifungal activities are related to the cationic nature of SLPI.SLPI distribution in vivo is mucosa-associated.It is synthesized and secretedin several glandular organs including the lung, mainly expressed in respiratorytract and germinal epithelium and is present constitutively in most mucosalsecretion (hence its alternative name mucus proteinase inhibitor). SLPI is foundin various secretory fluids including parotid secretions, bronchial, nasal, andcervical mucous, and seminal fluid. SLPI was originally isolated from parotidsaliva and has been detected in a variety secretions such as pancreatic secretion,whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears,and cerebral spinal fluid, as in secretions from the nose and bronchi, etc. SLPI isan important component of the antiprotease defense of tissues bathed bysecretory fluids and could be useful as a therapeutic agent for treatment of theproteolytic damage of tissues seen in degenerative and inflammatory diseases.SLPI protects the tissues by inhibiting the proteases, such as cathepsin G,elastase, and trypsin from neutrophils;chymotrypsin and trypsin from pancreaticacinar cells;and chymase and tryptase from mast cells. The SLPI gene wasfound to be expressed in lung, breast, oropharyngeal, bladder, endometrial,ovarian, and colorectal carcinomas, and SLPI detection is correlated with poorprognosis. SLPI is also found in neurons and astrocytes in the ischemic braintissue. Finally, SLPI was found to play a pivotal role in apoptosis and woundhealing.To date, hSLPI has mainly been extracted from tissues/cells or expressed inE.coli through gene engineering. But these methods have some insurmountableflaws,which confine the application of SLPI on a large scale. The origin oftissues/cells for protein extraction is limited and it is difficult in purification, andprone to cross contamination. As a host E.coli. has shortcomings such asendotoxin generating, plasmid liable to be lost, and so on, which make it neversurpass eukaryotic expression system. The foreign gene expression in E.coli. isinfluenced a lot. The cationic property of SLPI may bind to mRNA and DNA asa possible cause of toxicity to E.coli., which makes it impossible to be expressedat high level in E.coli. Adenovirus mediated gene therapy also has potentialdeficiency:(1) lack of target-specific action;(2) unable to be integrated into thehost DNA, leading to the short term of expression;(3) intense immune reactionand cell toxicity, which may be lethal to the host.According to the mentioned problems,we chose the Pichia pastoris to expressrecombinant protein. Pichia pastoris is unicellular eukaryotic organism, and it iscommonly used for expression recombinant protein in recent years. The benefitsof expression recombinant protein base on the following merits: the strongalcohol oxidase (AOX) promoter can control the foreign gene to express strictly;it can be cultureed with high density but require low nourishment which isbeneficial for industrial manufacture;it has very few self secrete protein and dogood for purification of foreign protein;the foreign gene is stable when it istransformed to the chromosome of the Pichia pastoris;it can process anddecorate the expression protein by itself, methylotrophic yeasts have the abilityto use methanol as a sole source of carbon and energy. Up until now expressionof SLPI in Pichia pastoris has not been found in any reports.In order to express human recombinant SLPI in Pichia pastoris we have donesuch work as follows:The construction of human recombinant SLPI expression system1. The construction of cloning vector contains HER2/neu ECD geneSLPI cDNA was cloned from Hella cell (SLPI over-expression in humancervical carcinoma cell line HeLa) through reverse transcription polymerasechain reaction (RT-PCR 5' primer5'-TCACTGCAGCCTTCACCATGAAGTCCAG, 3' prime5'-CTCCTCGAGATGGCAGGAATCAAGCTTTC). The SLPI DNA wassubcloned into a PBS clone plasmid. The recombinant vector was identified bydigestion of restriction enzyme PstI and XhoI. The result showed that the newrecombinant SLPI cloning vector was constructed successfully. DNAsequencing results showed that the cloning vector contained the complete genesequence of SLPI.2. The construction of expression vector contained SLPI geneTaking the cloning plasmid pBS-SLPI as the template, PCR (5' primer 5'-TGACTCGAGAAGAGATCTGGAAAGTCCTTC, 3' primer 5'-CGGTCTAGATTAAGCTTTCACAGGGGAAAC) amplified the DNA of theinterest gene. We constructed an expression plasmid pPICZα comprising SLPIgene. The recombinant vector was identified by the digestion of restrictionenzyme XhoI and XbaI. The result showed that the new recombinant SLPIexpression vector was constructed successfully. DNA sequencing results showthat the expression plasmid pPICZα-SLPI contained the correct reading frame.3. Transformation of the recombinant plasmid, identification of the positiveclones and the expression of the recombinant protein.Mixed 80ul of the fresh competence of yeast cells with 5～10μg of linearizedpPICZα-SLPI (in 5～10μl sterile water) and transfered them to an ice-cold(0℃) 0.2cm electroporation cuvette. Spread 50～100μl of each on separate, andlabeled YPD plates containing (25μg/ml) of Zeocin. To incubate plates for 2 to3 days at 28℃ until colonies formed. Positive transformants were selected andcultured in BMGY separately. When the Pichia pastoris were cultured in thelogarithmic phase , took 1ml culture liquid to extract the genome DNA of Pichiapastoris. And the SLPI transformants were assayed via PCR withno-transformant as antitheses. The result showed all transformants have theDNA fragment of 340bp but the antitheses has no DNA fragment of the interest.The Pichia pastoris in the logarithmic phase were centrifuged by1500r/minfor 5min. To resuspend the Pichia pastoris in the culture medium of BMMY andadd methanol with the concentration of 0.5%. It was ready to express of thegene of interest.The transformants were selected and induced by 0.5% methanol in BMMYmedium. The transformants were moved to orbital shaker in 28℃, with225r/min, and shaked to express.4. Analysis of the human recombinant SLPI proteinTook 20ul cultured supernatant of the 72h, which have been condensed byacetone for 2 to 12 times. The recombinant of the SLPI protein was detected bythe 15% SDS-PAGE and a protein band can be seen in the zone of 12kDa.For the first time SLPI protein was expressed in Pichia pastoris and thisprovides a basis for the developing research.