Studies On Expression Of RhuPA₁₇-KPI In Pichia Pastoris And Its Large-scale Fermentation Process

The urokinase-type plasminogen activator (uPA) is a kind of serine protease. It exists first in the form of single-chain urokinase-type plasminogen (pro-uPA); after catalyzed by plasmin, cathepsin, etc., the broken peptide bond K158-I159 forms chain A and chain B, becoming activated double-chain uPA. The chain A of uPA contains connecting locations with urokinase-type plasminogen activator receptor (uPAR); after effective combination between uPA and uPAR on the surface of cell, the fibrinolytic system is activated, the extracellular matrix and basilar membrane are degraded, and the signal transmission function inside the cell is activated. The amyloidβ-protein precursor contains a domain homologous to Kunitz-type serine protease inhibitors. The sequence of hKD/APP which is consisted of 57 amino acids has 43% sequence identity with that of BPTI. The spatial conformation, reactive center of KPI and its mechanism of function is much similar to that of BPTI. The hKD/APP show great ability to inhibit a variety of serine proteases such as factor XIa, epidermal growth factor(EGF)-binding protein, theγ-subunit of nerve growth factor, especially the inhibition constant KPI to factor XIa is higher than that of BPTI. The KPI has activity of growth factor and is an effective cell adhesion molecule. They probably play a role in the complex series of events that lead to tissue repair at vascular wound sites and protect neurocytes against injury. But the pathological and physiological role of this protease inhibitor domain which concerns with abnormal metabolism of APP are not well understood. Therefore we need to produce a large quantity of KPI to discover and study the possible mechanisms on pharmacodynamics. The bovine pancreatic typsin inhibitor is a representative inhibitor of Kunitz family. It inhibits trypsin with 1:1 stoichiometry. BPTI is a nonspecific and broad-spectrum serine protease inhibitor which affects known serine proteases such as trypsin, chymotrypsin, plasmin and kallikrein. It can defend blood platelet against bleeding, treat the patient with cruor impediment, in which situation the hemorrhage may occur. BPTI can be used in cardiac surgery as being extracorporal circulated to decrease the incidence of bleeding and the inflammatory reaction caused by the surgery. n order to improve these situations, studies were done as follows:①we constructed expression vector rhuPA17-KPI-pPICZαC and transformed it into Pichia pastoris via electroporation. The rhuPA17-KPI secreted into the culture supernatant at a high level and the Pichia pastoris was screened.②The rhuPA17-KPI was expressed in 80 L fermentor with optimizing parameters on large scale and creating a new method to purify rhuPA17-KPI.1. Screening and identification of stable and effective secretion expression rhuPA17-KPI pichia pastoris strain1)Build the rhuPA17-KPI-pPICZαC eukaryon expression carrier, recombine the PCR product to plasmid pPICZαC with same enzyme cutting; after corresponding enzyme cutting identification and sequencing proving, linearize the recombinant carrier and transform the pichia pastoris strain X-33.2)Screening of recombinant pichia pastoris strain:Apply the transformed pichia pastoris onto the YPD agar plate with Zeocin; after 72h cultivation under 28℃, select the clone of transforming yeast with Zeocin resistance, extract its genome DNA as template, and perform the identification with expression primer as PCR.3)Expression and identification of rhuPA17-KPITake the positive transformant after PCR identification, cultivate and amplify in shaker under 28℃, induce the expression with 0.5% methanol, after continuous separation of fermentation supernatant solution through centrifuge, the components at SDS-PAGE Commassie Blue Fast Staining single band are obtained. The supernatant protein of fermentation broth is analyzed.2.Establishment of large-scale fermentation and purification processes of rhuPA17-KPIThe pichia pastoris is a kind of single-cell eukaryote, as the expression host; with its inherent advantages, it is very suitable to produce exogenous protein by large-scale fermentation. This research explores the process of large-scale rhuPA17-KPI fermentation expression with 80 L fermenter. It emphasizes the factors significantly influencing the fermentation, such as pH value of fermentation broth, components of fermentation medium, Dissolved Oxygen degree (DO value), methanol replenishment speed, initial biomass, fermentation time, etc.Based on results, the optimization conditions are: FM21 fermentation medium with 0.5% peptone, pH5.5, induce the expression at about 190g /L of thallus density, the final methanol replenishment speed is 8.0~9.0ml/h/L volume of initial broth, DO 25~30%. the content of rhuPA17-KPI in broth can be 300mg/L. After large-volume continuous centrifuge, cation exchange chromatography and reversed water partition column chromatography and low-temperature rotary evaporation, rhuPA17-KPI can be 180mg/L(Bradford),over 95% in purity is obtained from supernatant, and the recovery rate is 60%.