Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties.

The present study reports the cloning and sequencing of lac2 from Bacillus subtilis. The gene is composed of 1542 bp and encodes a 514-amino acid protein. The gene has 86% homology with a published laccase with GeneID 936023. The lac2 gene was deposited in GenBank as a new nucleotide sequence. This new sequence was cloned into the multiple cloning site of pPIC9K to generate pPIC9K-lac2, which was then transformed into Pichia pastoris GS115 via electroporation. The recombinant GS115 (pPIC9K-lac2) was grown initially in BMGY medium and transferred to BMMY to induce gene expression for 48 h. The recombinant Lac2 protein shows laccase activity with α-naphthol and guaiacol as substrates. The optimal pH is between 3.2 and 4.7, and the optimal temperature is 25°C for enzyme reaction.

Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture.

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).

Recent advances on the GAP promoter derived expression system of Pichia pastoris.

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review.

[Constitutive expression of human angiostatin in Pichia pastoris using glycerol as only carbon source].

Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 (pGAP9K-AS)in shaking flask. The result shows that oleic acid is the best (163 mg/L) compared with glycerol (83mg/L), glucose (76 mg/L)and methanol (57 mg/L). Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 (pGAP9K-AS) in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P < 0.01).

[Construction of an inducible and efficient expression-secretion shuttle vector of B. subtilis].

A new shuttle vector pSB which could replicate in both Escherichia coli and Bacillus subtilis was constructed by fusing the E. coli plasmid pSP72 with the B. subtilis plasmid pUB18. After the sacB promoter and signal peptide sequence of B. subtilis was inserted in the multiple cloning sites (MCS) of pSB, The recombinant plasmid was designated as pSBP. The alpha-amylase gene terminator of Bacillus licheniformis was inserted at the other end of the MCS, resulting in expression-secretion plasmid pSBPT. After a positive control gene degQ of Bacillus pumilus was then cloned into pSBPT, and the inducible and efficient expression-secretion shuttle vector pSBPTQ was thus constructed. To identify the function and the necessary of sacB p. s., alpha-amylase terminator, and degQ in the expression of heterologous gene of pSBPT, the DNA fragment encoding for vasostatin I was cloned downstream of sacB p. s. of pSBP, pSBPT and pSBPTQ, and the resultant plasmid pSV, pSVT and pSVTQ were then transformed into B. subtilis strain DB1342. The transformants were screened on LB plates containing 10 microg/mL kanamycin. The positive transformants were separately grown on kanamycin containing 2 x MSR medium and sucrose was added to 2% final concentration for induction after 2h cultivation. The culture supernatant was used to run SDS-PAGE and Western blot. The results show that after induced by sucrose, very few recombinant Vasostatin I was expressed in DB1342(pSV), and recombinant Vasostatin I expressed in DB1342(pSVTQ) with the positive control gene degQ is more than in DB1342(pSVT) without degQ, suggesting that the Vasostatin I gene expression in pSVTQ was enhanced by degQ. Comparing the recombinant Vasostatin I in DB1342(pSVTQ) cells and its culture supernatant, the SDS-PAGE result show that most of the recombinant Vasostatin I was secreted into the culture supernatant and there is no Vasostatin in inclusion body, the secretion rate is about 90%. The result of plasmid stability test show that pSBPTQ maintains at 83% in B. subtilis after 40 generations.

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library.

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.

[Expression of human tumstatin in Pichia pastoris and its bioactivity].

Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.

Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter.

The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter (P(AOX1)) on pPIC9K resulting in plasmid pGAP9K. The recombinant expression vector pGAP9K-AS was constructed by inserting the angiostatin gene(AS) into pGAP9K. pGAP9K-AS was then transformed into P. pastoris GS115. The multi-copy integration transformant P. pastoris GS115 (pGAP9K-AS) was used to investigate the constitutive expression of angiostatin in P. pastoris. The expression of angiostatin reached its peak after 4 d of culture in P. pastoris GS115 (pGAP9K-AS) while the angiostatin expressed in P. pastoris GS115 (pPIC9K-AS) after 4 d of induction or 5 d of culture is only 70% of that expressed by P. pastoris GS115 (pGAP9K-AS). The AS expression in inducible system reached the peak after 6 d of induction but the expressed AS was only 86% of that from constitutive system. The results of anti-angiogenic and antitumor activity assay showed that AS expressed from both constitutive and inducible system inhibited the CAM angiogenesis and suppressed B16 melanoma in C57BL/6J mouse and that the tumor inhibition rates reached 90.63% and 90.54%, respectively. The above data indicates that the constitutive promoter P(GAP) can served as an effective alternative to the inductive promoter P(AOX1) to express AS and other proteins in P. pastoris.

The C-terminal domain of canstatin suppresses in vivo tumor growth associated with proliferation of endothelial cells.

Angiogenesis is crucial for the growth and metastasis of solid tumors with sizes larger than a few cubic millimeter Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was previously shown to inhibit proliferation of endothelial cells in vitro and suppress in vivo tumor growth. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in vivo, potently induced apoptosis of endothelial cells in vitro, and suppressed in vivo tumor growth in BALB/c mice. In the present study, we demonstrated that canstatin-C, the C-terminal 157-227 amino acid fragment of canstatin, also specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells and induced apoptosis, but the apoptosis-inducing activity, while close to that of the full-length canstatin, was much lower than that of canstatin-N. Canstatin-C also suppressed in vivo tumor growth in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-C is an anti-angiogenic domain of canstatin mainly associated with the specific inhibition of proliferation of endothelial cells, whereas canstatin-N with the potential apoptosis-inducing activity on endothelial cells.

Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth.

Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis. Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay. In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis. The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment. Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth.

[Expression and purification of the N-domain of human canstatin and its bioactivity].

Total RNA was extracted from placenta umbilical tissue. The canstatin cDNA was amplified from total RNA by net-RT-PCR technique and cloned into pSP72, and the resulted plasmid pSP72C was used as template to amplify its N-domain. The amplified 1-89 aa N-domain was then cloned into pET-3c. The resulted plasmid pET-CN was transformed into E. coli BL21(DE3). The N-domain was efficiently expressed after IPTG induction as a 10 kD band on SDS-PAGE. The expressed product accounted for approximately 35.3% of the total bacterial proteins, as estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed and the reactivated proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6%. CAM assay showed that N-domain effectively inhibited the angiogenesis of chicken embryo microcapillary vessel.

[Cloning and expression of kringle 1-3 gene of human plasminogen and the purification and bioactivity of its expressed product].

Kringle 1-3 domain is a recently found angiogenesis inhibitor with anti-angiogenesis and anti-tumor activity. The kringle 1-3 gene was amplified by PCR technique using angiostatin gene as template. After DNA sequencing, the PCR product was cloned into pPIC9K resulting in recombinant plasmid pPIC9K13 which was then transformed into Pichia pastoris GS115. The high copy integration transformants screened by PCR and G418 methods were cultivated in flasks. The K1-3 was expressed and secreted to the medium and has immunogenic activity as shown by SDS-PAGE and Western blotting. High cell density culture was carried out in 30-liter and 80-liter bioreactor, the biomass reaches 300 OD after methanol induction, and the expressed product is 200 mg/L. The fermentation supernatant was purified by Streamline SP and Phenyl Sepharose Chromatography, the product appears as a single band on SDS-PAGE, with a purity of 95%-96%. The purified product has anti-angiogenesis and anti-tumor activity.