Expression Of R-Elafin In Pichia Pastoris And Studies On Its Large-scale Fermentation And Purification Process

Elafin is an epidermal serine proteinase inhibitor, also known asskin-derived anti leukoproteinase (SKALP) or Elastase-SpecificInhibitor(ESI). It is first purified from secretions of patients with chronicobstructive pulmonary disease and cystic fibrosis. This inhibitor is provedto be an acid stable basic peptide with an isoelectric point of 9.7. Thecomplete amino acid sequence appears to be unique with 38% homology tothe C-terminal half of anti leukoprotease. The sequence shows that theinhibitor is composed of 57 amino acids and predicts a Mr of 6kD, it is alow-molecular-weight elastase inhibitor. The high affinity as well as theapparent specificity for elastases suggests a functional role in preventingelastase-mediated tissue proteolysis. So it can regulate the process of theorganism effectually. Elafin ,a serine elastase inhibitor,which inhibits the activity of serineproteinase inhibitor, regulates the release of the inflammatory cytokines,reduces the infiltration of the inflammatory cells and the release of toxicmatter in tissue. It is shown to be active against Gram-positive andGram-negative bacteria. In vein grafts, it is effective in reducing the earlyinflammatory response. And it attenuates post-cardiac transplant coronaryarteriopathy and reduces myocardial necrosis in rabbits after heterotopiccardiac transplantation. Additionally, it is very important in therapy ofvarieties of acute pathologies such as pneumonia, the acute respiratorydistress syndrome, myocardial infarction and psoriasis.Elafin can be expressed in many epithelial tissues such as folliculosis,oesophagus vagina and nonnasality etc. It is an epithelial host-defenseprotein that is absent in normal skin but highly induces in keratinocytes ofinflamed skin, psoriasis and after wounding etc. It is reported that tumournecrosis factor-alpha (TNF-α) and IL-1beta have been shown to stimulatethe production of elafin.Recently, we are interested in studies on Elafin by genic engineeringtechnology. Elafin can be expressed using many expression systems such asPichia pastoris and adenovirus expression systems. In addition, it issuccessfully expressed in transgenic mice. Studies were done as follows:①Cloning of elafin gene and construction of cloning plasmid pBKS-elafin;②Constructed expression plasmid pPICZαC-elafin and transformed it intoPichia pastoris via electroporation.We obtained r-Elafin secreted into theculture supernatant by the Pichia pastoris;③Studied the large-scalefermentation process of r-Elafin in the New Brunswick Scientific bioflow5000 fermentor;④Created a new method to purify r-Elafin.1. Cloning of elafin gene and construction of cloning plasmidpBKS-elafinWe first obtained the DNA sequence encoding Elafin by PCR usingspecific cloning primers from human genome. Then the purified PCRproduction was inserted into pBKS vector. It showed that the recombinantcloning vector pBKS -elafin was constructed correctly after identificationby endonuclease digestation assay and DNA sequencing.2. Construction of Pichia pastoris expression system for r-Elafin(1)With the recombinant plasmid of pBKS -elafin as template, weperformed PCR using expression primers with Xho I and Xba I target sitesand inserted the PCR products into pPICZαC. After confirmation byendonuclease digestation assay and sequencing, linearized the expressionvector pPICZ α C-elafin and transformed it into Pichia pastoris viaelectroporation.(2) Screening of the transformed Pichia pastoris: Extracted thegenomic DNA of the transformed yeasts to perform PCR using theexpression primers.(3) Expression and assay of r-Elafin: Proliferated the PCR testedpositively yeast clones, then induced the expression of r-Elafin with 0.5%methanol in BMMY media. Analyzed the expression products by activitydetermination and SDS-PAGE. In conclusion, we obtained the protein witha Mr of 6kD and it could inhibited PPE.3. Studies on large-scale fermentation and purification process ofr-Elafin(1) Studies on large-scale fermentation process of r-Elafin: Pichiapastoris has many advantages as a kind of expression host, and it is verysuitable for large-scale expression of the extraneous proteins. Accordingly,it is very important to study on large-scale fermentation of r-Elafin. So afteridentifying the bioactivities of the r-Elafin, we explored the large-scalefermentation process of r-Elafin and found that the best pH was pH5.2, DObetween 20%～30% and the supplied speed of methanol was 7.2ml/h/Linitial fermentation volume. The concentration of r-Elafin in the broth canreached 50mg· L-1 .(2) A new method to purify r-Elafin at large-scale: Supernatant offermentation was first purified by SP Sepharose XL with pH4.0.Then theeluate was performed onto Source 30RPC. R-Elafin was further purified ona mono S_ column HR 5/5 on pH4.0 for two times until approximatelyhomogeneity was observed.And the degree of purity could be more than95% and the yield was higher than 40%.In conclusion, our studies on r-Elafin had very important meaning,which established significant foundation for the further study.