Studies On Production Of Recombinant Human Bikunin And Its Inhibitory Effects On Tumor Invasion And Metastasis

Bikunin is a proteoglycan containing two Kunitz-type proteases inhibitor domains. It has a molecular mass of about 25 kDa and carries a 6 to 7-kDa chondroitin sulfate chain. Bikunin exhibits broad-spectrum inhibitory activity against serine proteases such as trypsin, chymotrypsin, leukocyte elastase, and plasmin. It may also reduce the production of inflammatory factors such as TNF, IL-6 and IL-8 and has been widely used as a drug for patients with acute inflammatory disorders such as pancreatitis, disseminated intravascular coagulation and shock. It also exhibits protective effect against ischemia- reperfusion injury and could potentially suppress tumor cell invasion and metastasis. In this study, we expressed recombinant human Bikunin (rhBikunin) with P. pastoris X-33, a wild type Pichia strain containing the AOX1 promoter which allows for rapid growth while utilizing methanol as the sole carbon source. After expressed in shake flask, rhBikunin was produced in an 80 L fermenter. A new large-scale purification procedure was established. The effects and the mechanism of recombinant and native human Bikunin on tumor invasion and metastasis were investigated in vitro and in vivo.1. Shake-flask expression of rhBikunin and selection of high-level expression colonies1.1 Construction of expression vector pPICZαC-bik Construction of the recombinant expression vector was based on pPICZαC plasmid. The insert containing cDNA encoding human Bikunin was prepared via PCR using the vector pMD18T-AMBP as the template. The upstream primer contains the recognition sequence for XhoⅠand coding sequences for the KEX2 protease recognition sequence (Lys–Arg). The downstream primer contains the recognition sequence for XbaⅠ. The nucleotide sequences of the cDNA insert and flanking sequence were verified by sequencing. Results of DNA sequence analysis of the recombinant expression vector pPICZαC-bik demonstrated that cDNA encoding human bikunin was correctly inserted into pPICZαC vector and the sequence was consistent with that logged in GenBank (accession no. 001633).1.2 Transformation of Pichia pastoris and selection of high-level expression coloniesThe linearized recombinant expression vectors were introduced into P. pastoris X-33 by electroporation using a Micropulser. pPICZαC blank plasmids were also transformed as a negative control. Transformed cells were selected by growth on yeast extract peptone dextrose (YPD) agar plates containing zeocin. Antibiotic Zeocin was used in two concentrations: 0.1 and 0.2 mg/mL. After the multicopy transformants appeared, Twelve colonies were cultured in shake flask and the expression level of rhBikunin was evaluated by TIA analysis of expression medium supernatant per day over 7 days induction by methanol with pH 6.0. Levels of rhBikunin increased gradually after induction and reached maximum at day 3, then began to decrease. The maximum level of rhBikunin of clone 3, marked as X33-BIK03, was ~120 IU/mL, higher than that of other clones. For clone 12 which was transformed with pPICZαC blank plasmid, the level of recombinant bikunin was undetectable.In order to ascertain whether cDNA encoding bikunin was integrated into the P. pastoris host cell genome, PCR was performed on the transformed yeast genomic DNA. Agarose gel electrophoresis of the PCR products showed that cDNA encoding bikunin was indeed integrated in all 11 clones transformed with bikunin recombinant expression vector pPICZαC-bik. There were no visible bands from the control sample which were transformed with pPICZαC blank plasmid.The expression levels of rhBikunin were different when the pH of medium varied from 3.0 to 7.0. TIA reached a maximum (~210 IU/mL) when the pH of the medium was 4.0.2. Characterization of rhBikunin expressed in P. pastoris According to SDS-PAGE analysis, rhBikunin migrated as a double band.Their molecular masses based on protein migration rates in SDS gel were 24.2 kDa and 20.8 kDa respectively. Both forms were shown to be immunoreactive by Western blotting analysis. N-terminal sequencing of the 24- and 21-kDa proteins suggested that these proteins was identical to the N-terminus of native human Bikunin (accession no. CAA38585), demonstrating correct signal peptide processing. No carbohydrate side-chains could be detected in recombinant human bikunin with chondroitin ABC lyase and N-glycosidase F.3. Pilot-scale expression and purification of rhBikuninA stock culture of recombinant P. pastoris X33-BIK03 was grown to an A600 of 6.0 in a 5 L shake flask containing 2 L YPD. The shake-flask culture was used to inoculate a 80 L fermenter containing 40 L of fermentation basal salts medium FM21 supplemented with PTM1 trace salts and biotin. The glycerol phase lasted 25 h and the methanol feed started when the culture reached an A600 of 140 and wet weight of 191 g/L. Under optimal conditions (pH 4.0, 28℃, and DO set at 20-30%), an A600 of 329 (corresponding to a wet weight of 245 g/L) was obtained after 60 h of induction. Recombinant Bikunin was secreted in the medium after induction and a final concentration of approximately 320 IU/mL was obtained.The rhBikunin solution was purified with cation exchange chromatography (SP Sepharose XL) and reverse phase chromatography (Source 30). With an AKTA Explorer 100 chromatography system, the optimal concentration of NaCl for elution was 0.5 M. Following these processes, we could get totally 1.44 g of purified rhBikunin from 40 L culture medium, the purity of rhBikunin reached 94.7% as revealed by SDS-PAGE. The specific activity of rhBikunin was 5600 IU/mg, which was 2.24 times higher than that of native human urinary bikunin.4. Effects of rhBikunin on invasion and migration of tumor cells in vitro MTT assay was used to examine effects of rhBikunin on growth and proliferation of HO8910 PM cells. Effects of rhBikunin on adhesive ability of HO8910 PM cells was tested by cell-fibronectin adhesion assay. Millicell chamber assay was performed to determine effects of rhBikunin on invasion and migration capacities of HO8910 PM cells. The results demonstrated that, rhBikunin could promote cells'adhesion to fibronectin and inhibit cells's invasion across the Matrigel basement membrane. Migration potentials of HO 8910 PM cells were not influnced significantly by recombinant or native Bikunin .5. Effects of rhBikunin on tumor invasion and metastasis in vivo C57BL/6 male mice were inoculated with Lewis lung carcinoma cells (2×106) into the subcutaneous. From the second day, intraperitoneal administration of NaCl, rhBikunin (30 000, 60 000, 120 000 IU/kg), native Bikunin (Wusitading, 120 000 IU/kg) was repeated daily for 20 days. Autopsy was performed at the 21st day. Primary tumors and lungs were collected and fixed in 10% neutral formalin buffer. Then sections stained by HE were made.High dose of rhBikunin, as well as Wusitading, caused a significant reduction in tumor volume and weight. Pathohistological examination of primary lesion showed that, carcinoma cells have infiltrated into muscle or periosteum in control group and low dose of rhBikunin-treated group. While in high dose of rhBikunin- treated or Wusitading-treated group, carcinoma cells remained in situ. The incidences of pulmonary metastasis of Lewis lung carcinoma were both 25% in high dose of rhBikunin-treated group and Wusitading-treated group, much lower than that in control group, which was 100%. All the results suggested that, rhBikunin, the same as native Bikunin, has great potential in clinical treatment of tumor metastasis.6. Effects of rhBikunin on mRNA and protein expression of uPA in tumor cells RT-PCR results demonstrated that, the mRNA expression of uPA in HO8910 PM cells was down-regulated by recombinant and native Bikunin. As results of SABC immuneohistochemical method, positive expression of uPA was fewer in high dose of rhBikunin-treated group or Wusitading-treated group, compared with control group and low dose of rhBikunin-treated group. The antimetastasis effects of Bikunin might be partly resulted from down-regulation of uPA expression in cancer cells, as well as the inhibitory activity against uPA and plasmin, which could prohibit the degradation of extracelluar matrix.Our studies provied a new approach of production of highly purified rhBikunin. The recombinant Bikunin was proved to have the same functions as the native Bikunin.