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Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirus-infected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37 degrees C, but increased to 85% at 22 degrees C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBP-fused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.

In this study, three-dimensional rotordynamic analyses have been conducted using equivalent beam, hybrid and full three-dimensional models. The present computational method is based on the general finite element method with rotating gyroscopic effects of the rotor system. General purpose commercial finite element code, SAMCEF which includes practical rotordynamics module with various types of rotor analysis tools and bearing elements is applied. For the purpose of numerical verification, comparison study for a benchmark rotor model with support bearings is performed first. Detailed finite element models based on three different modeling concepts are constructed and then computational analyses are conducted for the realistic and complex three-dimensional rotor system. The results for rotor stability and mass unbalance response are presented and compared with the experimental vibration test data conducted herein.

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeastHansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase ( (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using theMOX promoter ofH. polymorpha and the mating factor α secretion signal ofS. cerevisiae. Multiple integrants of a transformang vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression inH. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.

A novel process using O2-enriched air supply was used to suppress the C-terminal proteolytic degradation of recombinant hirudin (r-hirudin) from Saccharomyces cerevisiae. When dissolved O2 was controlled above 20% saturation level using normal air, inactive forms of C-terminally truncated hirudin were observed in culture broth from 48 h of fermentation. The use of O2-enriched air giving above 40% saturation of dissolved O2 suppressed the proteolytic degradation and hence the formation of truncated forms of inactive r-hirudin until 60 h of fermentation.

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1alpha (stromal cell-derived factor-1alpha) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1alpha fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP-RANTES and MBP-SDF-1alpha was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1alpha.

This study examined the possibility of an adaptive reaction of anastomosed arteries under tension during a distraction lengthening procedure in the tibiae of rabbits. After an osteotomy at the mid tibia, the posterior tibial arteries were transected and anastomosed. Using a pair of small external fixators, the tibiae were distracted at a rate of 0.5mm/day (groups I-IV rabbits). Three weeks after 25% lengthening, the patency and histology of the arteries were examined. Angiography revealed that all of the anastomosed arteries were patent, and intimal hyperplasia was a constant finding. The mean thickness of the intima of the lengthened segment in group I was 60.4 microm, which is 5.0, 3.4 and 2.1 times higher that of the controls in groups IV (un-manipulated arteries, 12.2 microm), III (unlengthened but anastomosed arteries, 17.8 microm) and II (lengthened but untouched arteries, 28.7 microm), respectively. These results show that an anastomosed artery can maintain its patency at a certain level and speed of distraction lengthening. Therefore, it is possible that distraction lengthening and vascular anastomoses can be performed simultaneously provided there is careful monitoring of the circulation.

Streptomyces exfoliatus SMF19 produced two types of extracellular beta-lactamase-inhibiting proteins, 48 (beta LIP-I) and 33 (beta LIP-II) kDa. The inhibition mode of beta LIP-I on Bacto Penase and benzylpenicillin as substrates was uncompetitive (i.e., both Km and Vmax were changed), while that of beta LIP-II was noncompetitive (i.e., only Vmax was changed). The inhibition constants of beta LIP-I and beta LIP-II were 0.62 x 10(-4) and 2.74 x 10(-1) mumol, respectively. The N-terminal amino acid sequence of beta LIP-I was NH3-*-S-T-V-F-D-L-V-*-L-G, and that of beta LIP-II was NH3-D-F-*-V-F-D-L-E-A-T-D-E.