• Journal of Microbiology
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• v.45 no.6
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• pp.572-577
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• 2007

To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.2004-2007
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• 2006

An efficient method for the in vitro reassembly of homologous DNA sequences is presented. The proposed method involves obtaining single strands of homologous genes and hybridizing them to obtain partially hybridized heteroduplex DNA; cleaving the single-stranded regions of the heteroduplex DNA using S1 nuclease to generate double-strand DNA fragments; denaturing the double-strand DNA fragments to generate single-strand DNA fragments; conducting a series of polymerase chain reactions (PCR) using the single-strand DNA fragments as internal primers and a mixture of homologous DNA as templates to obtain elongated reassembled DNA; and finally, amplifying the reassembled DNA by a PCR using terminal primers. As a result, DNA reassembly could be achieved between homologous genes with a sequence similarity as low as 78%.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.764-769
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• 2000

The lipase gene(lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3 base changes, resulting in two cryptic mutations and one amino acid substitution: S113($AGC{\rightarrow}AGT$), L252 ($TTG{\rightarrow}TTA$), and G275E ($GGA{\rightarrow}GAA$). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity ($V_{max}$) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200 U/mg, which was 10% higher than that of the parental (WT) lipase (2,900 U/mg). Its optimum temperature for the hydrolysis of olive oil was $68^{\circ}C$ and it showed a typical $Ca^{2+}$-dependent thermostability, properties fo which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.

• The Korean Journal of Applied Statistics
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• v.23 no.1
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• pp.189-196
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• 2010

It is never an easy task to physically randomize the sequence of cards. For instance, US 1970 draft lottery resulted in a social turmoil since the outcome sequence of 366 birthday numbers showed a significant relationship with the input order (Wikipedia, "Draft Lottery 1969", Retrieved 2009/05/01). We are motivated by Laplace's 1825 book titled Philosophical Essay on Probabilities that says "Suppose that the numbers 1, 2, ..., 100 are placed, according to their natural ordering, in an urn, and suppose further that, after having shaken the urn, to shuffle the numbers, one draws one number. It is clear that if the shuffling has been properly done, each number will have the same chance of being drawn. But if we fear that there are small differences between them depending on the order in which the numbers were put into the urn, we can decrease these differences considerably by placing these numbers in a second urn in the order in which they are drawn from the first urn, and then shaking the second urn to shuffle the numbers. These differences, already imperceptible in the second urn, would be diminished more and more by using a third urn, a fourth urn, &c." (translated by Andrew 1. Dale, 1995, Springer. pp. 35-36). Laplace foresaw what would happen to us in 150 years later, and, even more, suggested the possible tool to handle the problem. But he did omit the detailed arguments for the solution. Thus we would like to write the supplement in modern terms for Laplace in this research note. We formulate the problem with a lottery box model, to which Markov chain theory can be applied. By applying Markov chains repeatedly, one expects the uniform distribution on k states as stationary distribution. Additionally, we show that the probability of even-number of successes in binomial distribution with trials and the success probability $\theta$ approaches to 0.5, as n increases to infinity. Our theory is illustrated to the cases of truncated geometric distribution and the US 1970 draft lottery.