| The humanization, high efficacy and miniaturization of antibody as well as the seeking of novel molecular targets are topics of the mainstream in the development of antibody-based drugs. Antibody-directed drugs can orient some biologically active substances to the target sites in tumors by means of the specific binding between the antibody moiety and its corresponding antigen and finally exhibit antitumor effects. As a worthwhile and molecular target for the antitumor study, typeⅣcollagenase is highly expressed in a variety of human solid tumors. Lidamycin (LDM), a member of the enediyne antibiotic family, displays extremely potent cytotoxicity toward cancer cells and exerts unique DNA cleavage. The LDM molecule consists of two moieties, an enediyne and an apoprotein (LDP), which can be separated and reconstitued. At present, LDM has finished its phase I clinical trial. The engineered fusion protein Fv-LDP was expressed as inclusion body in the recombinant E.coli BL21(DE3)StarTW/pEFL. E.coli BL21(DE3)StarTM/pEFL was constructed by the transformation of host strain E.coli BL21(DE3)StarTM using recombinant plasmid pEFL carrying the fusion gene Fv-LDP which consisted of the single-chain Fv fragment derived from the anti-typeⅣcollagenase antibody and the apoprotein LDP. Energized fusion protein Fv-LDP-AE was obtained by adding the active enediyne (AE) of LDM into the molecule of fusion protein Fv-LDP. Therefore, the energized fusion protein Fv-LDP-AE is unique for its specific binding to typeⅣcollagenase and its extremely potent cytotoxicity to cancer cells. This paper includes two parts, the antitumor activity of energized fusion protein Fv-LDP-AE and the high-density cell cultivation of fusion protein Fv-LDP producing strain E.coli BL21(DE3)StarTM/pEFL.1. The antitumor activity of the energized fusion protein Fv-LDP-AEDNA damage assay showed that the energized fusion protein Fv-LDP-AE could damage DNA dose-dependently. The effective concentration of cutting plasmid by Fv-LDP-AE was 1.95×10-8M per 0.45μg DNA without the presence of reducer and that of LDM was 2.5×10-8M per 0.45μg DNA. Furthermore, there existed common smear bands for 1.5μg of the nude genome DNA treated by Fv-LDP-AE and LDM. Taken together, these data suggested that Fv-LDP-AE was similar to LDM in the mode of directly cutting DNA.Antitumor activity of Fv-LDP-AE against cancer cells was further observed by MTT assay. The IC50 values of Fv-LDP-AE for highly metastatic human giant cell lung carcinoma PG cells, low metastatic human lung adenocarcinoma PAa cells, and human hepatoma BEL-7402 cells were 1.47×10-10 M, 1.57×10-9 M, and 1.10×10-10 M, while those of LDM were 2.41×10-10 M, 2.91×10-9 M, and 1.61×10-10M, respectively. The results implicated that the antitumor activity of Fv-LDP-AE was more potent, in some degree, than that of LDM. This might be associated with the action of scFv in the energized fusion protein Fv-LDP-AE.With 0.01 nM, 0.1 nM, 0.5nM, 1 nM of Fv-LDP-AE or LDM for 4 h, followed by 72 h incubation in drug-free medium, the treated PG cell, PAa cells and BEL-7402 cells all displayed mitotic cell death characterized by enlargement of cell volume and cell nuclei, delayed G2+M phase and appearance of multinucleation. Hoechst 33342 and PI staining showed a unique and atypical chromatin condensation. The cells with chromatin condensation were characterized by nearly integrated karyotheca, appearance of small "dot" representing segregated condensed chromatin, and different morphology and frequency of multinucleated cells. BEL-7402 cells treated with 0.1 nM Fv-LDP-AE were easier to enter the mitotic cell death than those treated with 0.1 nM LDM. The former caused 71% of multinucleated cells and the latter 58 %, respectively. With the same treatment, both of the drugs at 0.1 nM could cause more than 40 % of PG cells to be multinucleated. However, the percentage of multinuCleation decreased at further higher drug concentration. No more than 34.8 % of multinucleated PAa cells were found and Fv-LDP-AE was easier to promote multinucleation in PAa cells. These findings suggested that the frequency of multinucleation was varied in different cell lines.Besides mitotic cell death, cell apoptosis also occurred in PG cells and BEL-7402 cells exposed to Fv-LDP-AE or LDM ranging from 0.1 to 1 nM for 4 h, followed by 72 h incubation in free drug medium. About 20 % or less of PG cells showed apoptotic when treated with Fv-LDP-AE or LDM. However, the DNA ladder was never observed in the control or the treated PG cells, which implied that all of the cells might be at the early phase of apoptosis. Flow cytometry analysis showed that BEL-7402 cells displayed typical and apoptotic sub-G1 peaks after treatment of Fv-LDP-AE and LDM compared with the control. Furthermore, the percentage of apoptosis diminished with drugs within the range of 0.1 nM and 1 nM. DNA ladder also appeared in treated BEL-7402 cells analyzed by agarose gel electrophoresis. No representative apoptosis occurred in the treated PAa cell.Fv-LDP-AE and LDM also affected the progress of cell cycle. Fv-LDP-AE and LDM at 0.1 nM caused 56.9 % and 60.7 % of PG cells to retard at G2+M phase, respectively. However, PG cells treated with 0.5 nM and 1 nM of Fv-LDP-AE or LDM were arrested at G2+M and S phase simultaneously. The maximal percentage of PG cells in S phase was 69.1% and 79.4 %, respectively. The analysis of cell cycle was not feasible because there were few of cells in the cycle due to the occurrence of apoptosis and mitotic cell death in the treated BEL-7402 cells.SA-β-gal staining revealed that some cells displayed senescence-like phenotype when treated with Fv-LDP-AE or LDM. Similar percentage of positive SA-β-gal was found in the same cell line after exposure to Fv-LDP-AE or LDM, which implied that both of them showed no difference in the induction cell senescence. In addition, telomerase activity in LDM-treated BEL-7402 cells was markedly decreased which might play a key role in mitotic cell death and cell aging morphology. The details need to be further investigated.2. High-density cell culture of recombinant E.coli BL21(DE3)StarTM/pEFL producing fusion protein Fv-LDP High-density cell cultivation is an effective way to improve the yield of biologically engineering product. The success of high-density cell cultivation is determined by manifold factors such as stability of strains, medium composition, strategies of fermentation, and so on.1) Stability monitoring on the expression of engineered fusion protein Fv-LDP in produdng strain E.coli BL21 (DE3) StarTM/pEFLAn engineered fusion protein Fv-LDP that composes the Fv fragment derived from mAb 3G11 directed against typeⅣcollagenase and the apoprotein of LDM, an antitumor antibiotic with extremely potent cytotoxicity to cancer cells has been prepared in this laboratory. Stability monitoring on the expression level in the producing strain E.coli BL21(DE3)StarTM/pEFL was a requirement for upscale preparation of fusion protein Fv-LDE The lyophilized stock-strain has been investigated for its physical, chemical, microbiological and biological characteristics at regular intervals. Gram staining and IMViC assays revealed that the strain was in accordance with the features of E. coli. Furthermore, the restriction enzymatic analysis of recombinant strain E.coli BL21(DE3)StarTM/pEFL was performed. The agarose electrophoresis indicated that enzyme EcoR I could cleave the recombinant plasmid into one 6.5 kb band while the Nde I and Xho I enzymes could digest the plasmid into two different bands corresponding to the size of free vector pET-30a(+) and Fv-LDP gene, respectively. Moreover, the sequencing result of Fv-LDP gene attained from the 100th generation of recombinant strain was uniform with the original recombinant strain. In addition, Western blot using anti-His-tag as the primary antibody made it clear that Fv-LDP was expressed stably in different generations of the recombinant strain. SDS-PAGE analysis discovered that the purity of Fv-LDP was more than 96% after the process of immobilized metal affinity chromatography. ELISA assay also suggested that fusion protein Fv-LDP in part retained the affinity of mAb 3Gll to the antigen typeⅣcollagenase that was secreted by human lung carcinoma PG cells with highly metastatic potential. Moreover, Fv-LDP itself could partially inhibit the activity of typeⅣcollagenase analyzed by gelatin-zymography. In conclusion, all the characteristics described as above were identical to the original recombinant strain. This indicated that E.coli BL21(DE3)StarTM/pEFL was relatively stable and suitable for the fermentation study on a pilot scale.2) The optimization of culture composition and culture conditions for the E.coli BL21 (DE3) StarTM/pEFL producing fusion protein Fv-LDPSpot Denso software in the Chemilmager gel imaging system is utilized to determine the relative quantity of the target protein. Based on this, a calibrated linear curve of integrated density value (IDV) versus concentration of fusion protein Fv-LDP was shown within 88.7-1064μg/mL (A = 222.7C, r = 0.9988). The recovery rate was 95.18 % with the RSD of 2.09 % (n = 8). These above-mentioned data suggested that the method was feasible in the quality control of fusion protein Fv-LDP.The composition of fermentation medium and culture conditions was optimized in shaking flask by single factor test and orthogonal test. After a series of selections, an optimal medium composition was obtained. It contains: homemade malt extract 4 g/L, tryptone 10 g/L, yeast extract 17.5 g/L, beef extract 17.5 g/L, ammonium sulfate 1g/L, disodium hydrogen phosphate 1.5 g/L, potassium dihydrogen phosphate 1.5 g/L, sodium chloride 2.5 g/L, and magnesium sulfate 1.5 mL/L. The optimization of culture conditions was performed using the optimal medium obtained. The optimal conditions beneficial to the growth of recombinant strain and expression of Fv-LDP are listed as follows:①the seed strains are cultured using the same fermentation medium;②the recombinant strains grow at 37℃and are induced at the same temperature;③the initiative pH value of fermentation medium is 7.5;④the inoculation volume is 6%;⑤the recombinant strains should grow for 6 h and be induced for the same time;⑥the final concentration of IPTG is 0.1 mM. Under these optimal conditions, fusion protein Fv-LDP was finally produced at the expression level of more than 800μg/mL in E.coli BL21 (DE3) StarTM/pEFL recombinant strains. The output of Fv-LDP was 6 to 7 times higher than that of LB, which suggested that the optimization of medium composition and culture conditions was necessary to improve the expression level of fusion protein Fv-LDP.3) The high-density cell cultivation of E.coli BL21 (DE3) StarTM/pEFL producing fusion protein Fv-LDPA great deal of foam came into being during the high-density cell cultivation of the engineered strain, so 0.2‰of the antifoam was added into the fermentation jar. The recombinant strain was first cultured in the fermentation jar under the conditions similar to that of shaking flask. The yield of fusion protein Fv-LDP was got at the level of 403.5μg/mL, lower than that of shaking flask, which might be associated to the limited dissolved oxygen. In order to increase the level of dissolved oxygen, the rotating speed of stirrer was set at 600 rpm in the batch fermentation of the recombinant strain. The increase in the expression level of fusion protein Fv-LDP (700μg/mL) remained to be unsatisfied. Therefore, fed-batch fermentation was tried for a big boost in yield of fusion protein Fv-LDP. In the pH-stat fed-batch fermentation, fusion protein Fv-LDP was expressed at about 3 g/L. However, the presence of high level glucose and amino nitrogen was critically detrimental to E.coli BL21(DE3)StarTMpEFL。In order to avoid the stress of glucose and amino nitrogen on the expression of fusion protein Fv-LDP, the stepwise and constant speed fed-batch fermentation was carried out to culture the recombinant strain. The optical density at 600 nm of fermented broth was enhanced time-dependently. Accordingly, was the accumulation of fusion protein Fv-LDR The maximal yield of Fv-LDP approximately reached 4 g/L. Glucose concentration was slightly reduced and finally maintained at the moderate level of 4 mg/mL during the whole cultivation. The level of amino nitrogen ever reduced to 15 mg per 100 milliliter. These data suggested that the medium composition was better assimilated and the stepwise and constant speed fed-batch fermentation was a good protocol in favor of the growth of E.coli BL21 (DE3)StarTM/pEFL and the expression of fusion protein Fv-LDP.
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