• KSBB Journal
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• 제14권4호
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• pp.482-489
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• 1999

As host for the production of eucaryotic heterologous proteins, methylotrophic yeast Pichia pastoris and Hansenula polymorpha are the most highly developed of a small group of alternative yeast species chosen for their perceived advantages. This paper describes the method to enhance the recombinant protein productivity with P. pastoris and H. Plymorpha. In these experiments, the effects of methanol induction timing, induction method, pH, culture temperature and kinds of nitrogen sources on foreign protein production were tested with P. pastoris and compared with H. polymorpha.. In addition, optimum methanol concentration as inducer and the effects of carbon sources on AOX1 or MOX promoter repression and secretion efficiency were also studied in both cases.

• BMB Reports
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• 제38권4호
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• pp.414-419
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• 2005

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권2호
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• pp.84-91
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• 2000

Mycopasma contamination of tissue culture cells easily evades detection and, thus, represents a continous therat to cell biologists. In case where infected cell can not simply be replaced, attempts have to be made to eradicate mycoplacma from the tissue culture cells. A variety of anti-microbial agents have been shown to be toxic to mycoplasma strains ; however, cell associated mycoplasma are often protected from antibiotics at concentrations shown to be effective in vitro. Antibiotic concentrations high enough to be lethal to cell as sociated mycoplasmas frequently are also detrimentrations to the host cells, while moderately increased antibiotic levels tolerated by the host cells often lead to only temporary growth suppression and/or to the emergence of mycoplasma strains resistanct even to high concentrations of the antibiotis applied. Hare, a genetic approach for the elimination of mycoplasma from tissue culture cells that overcomes thens limitations is described. By expression of a selection marker conferring resistance to an otherwise toxic agent, Acholeplasma laidlawii infected BHK-21 cells used as the model system were enabled to temporarily tolerate antibiotic concentrations high enough to be lethal to cell associated mycopalsma while leaving the host cells unharmed. Upon successful mycoplasma eradicated, cultvation of the cured host cells in the absence of the selective agent yielded revertant cell clones that had regained susceptibillity to the toxic agent. Cressation of the selection marker expression was shown to result from the loss of the selection marker DNA, which is a consequence of the fact that the stable and permanent integration of foreign DNA in eucaryotic cell chrosomes is highly inefficient. Thus, the cells were cured from mycoplasma yet remained biochemically unaltered.

• 생명과학회지
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• 제14권2호
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• pp.315-319
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• 2004

사람의 serine palmitoyltransferase(SPT, EC 2.3.1.50)에 대한 항체를 제작하기 위하여 E. coli발현 vector인 pRset vector에 SPTLC1 및 SPTLC2 유전자를 subcloning하고 BL21 (DE3)pLys cell에 발현시켰다. 포유동물의 SPT는 원핵세포의 SPT homodimer와는 달리 SPTLC1 및 SPTLC2 2개의 sub-unit로 된 heterodimer이다. Human embryo kidney cell인 HEK293 cell의 total RNA로부터 RT-PCR을 행하여 cDNA library를 얻은 다음 SPTLC1 및 SPTLC2의 특이적인 primer 들을 이용하여 PCR을 행하였다. SPTLC1 및 SPTLC2 DNA를 hexahistidine fusion 단백질을 발현시킬 수 있는 pRset vector에 cloning하여 pRsetB/SPTLC1 및 pRsetA/SPTLC2를 얻고 염기서열을 확인하였다. 재조합 plasmid를 발현세포인 BL21 cell에 형질전환시킨 다음 ampicillin 및 chroramphenicol 배지에서 선별하여 재조합세포를 얻었다. 1 mM IPTG로서 발현을 유도하였으며 세포 단백질을 SDS-PAGE로 분리한 다음 His-tag antibody로 western blotting을 행하여 SPTLC 및 SPTLC2가 발현되었음을 확인하였다.