Jin-Oh Baek

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.

Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography

The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to > 98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60 nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine.

Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer

We developed a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5-1.0 M NaCl. The HPV33 L1 protein was shown to be approximately 56 kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20-40 nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production as an antigen delivery system. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.

Heterologous Expression and Characterization of L-Amino Acid Deaminase from Proteus mirabilis in Escherichia coli

We expressed an L-amino acid deaminase (Pma) from Proteus mirabilis (P. mirabilis) in Escherichia coli and characterized the kinetics of phenylpyruvic acid production. P. mirabilis Pma was well expressed in E. coli in an active state and was found to be associated with membranes. The association of Pma with cellular membranes is likely to be necessary for its enzymatic activity.