• 제목/요약/키워드: family shuffling.

검색결과 6건 처리시간 0.031초

Gene Family Shuffling을 이용한 Cytidine Deaminase 활성 증가 (Enhanced Activity of Cytidine Deaminase by Gene Family Shuffling.)

  • 홍식;김경동;송방호;정경화;김사열
    • 한국미생물·생명공학회지
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    • 제30권4호
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    • pp.298-304
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    • 2002
  • PCR방법을 기본으로 한 in vitro recombination과 대장균 cdd 돌연변이주에서의 발현을 통하여 family shuffling이 수행되었다. 고온성 Bacillus caldolyticu와 B. stearothermophilus 유래의 시티딘 디아미나제을 코드하는 cdd 유전자를 shuffling하였다. 이것을 대장균 cdd 돌연변이주에 형질전환 시킨 후 uraci이 없는 AB배지에서의 생존을 통하여 150개의 돌연변이 균주를 얻을 수 있었으며, 그 중 연구를 위하여 4주(SH1067, SH1077, SH1086, 및 SH1118)를 선택하였다. 선택한 4주의 염기서열을 분석한 결과, 수 회의 point mutation과 recombination이 각각 일어났음을 확인 할 수 있었다. 특히 SH1067의 경우,$ 80^{\circ}C$에서 B. stearothemophilus 에서 유래한 7101의 시티딘 디아미나제 활성과 비교하여 770배 이상의 증가를 보여주었다.

Molecular Breeding of Genes, Pathways and Genomes by DNA Shuffing

  • Stemmer, Willem P.C.
    • Biotechnology and Bioprocess Engineering:BBE
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    • 제7권3호
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    • pp.121-129
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    • 2002
  • Existing methods for optimization of sequences by random mutagenesis generate libraries with a small number of mostly deleterious mutations, resulting in libraries containing a large fraction of non-functional clones that explore only a small part of sequence space. Large numbers of clones need to be screened to find the rare mutants with improvements. Library display formats are useful to screen very large libraries but impose screening limitations that limit the value of this approach for most commercial applications. By contrast, in both classical breeding and in DNA shuffling, natural diversity is permutated by homologous recombination, generating libraries of very high quality, from which improved clones can be identified with a small number of complex screens. Given that this small number of screens can be performed under the conditions of actual use of the product, commercially relevant improvements can be reliably obtained.

Effective Family Shuffling Method Using Complementary DNA Fragments Produced by S1 Nuclease

  • Hong, Soon-Gyu
    • Journal of Microbiology and Biotechnology
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    • 제16권12호
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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%.

DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability

  • Yao, Zhuang;Jeon, Hye Sung;Yoo, Ji Yeon;Kang, Yun Ji;Kim, Min Jae;Kim, Tae Jin;Kim, Jeong Hwan
    • Journal of Microbiology and Biotechnology
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    • 제32권6호
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    • pp.800-807
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    • 2022
  • Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

Guided Selection of Human Antibody Light Chains against TAG-72 Using a Phage Display Chain Shuffling Approach

  • Kim, Sang-Jick;Hong, Hyo-Jeong
    • Journal of Microbiology
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    • 제45권6호
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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.

새로운 유전자 재조합 방법을 이용한 대장균에서의 인간 tissue inhibitor of mtrix metalloproteinase-2 (TIMP-2) 유전자의 가용성 발현 (Enhancement of the solubility of human tissue inhibitor of matrix metallocroteinase-2 (TIMP-2) in E. coli using a modified in vitro mutagenesis)

  • 김종욱;최동순;주현;민철기
    • KSBB Journal
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    • 제23권3호
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    • pp.231-238
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    • 2008
  • 암세포의 침윤은 숙주 조직의의 기저막과 세포외 기질을 침투함으로써 일어난다. 침윤과 전이과정에는 단백질가수분해 효소인 matrix metalloproteinases (MMPs)가 깊이 연관되어 있는 것으로 알려져 있으며, MMP의 가수분해 활성은 tissue inhibitors of metalloproteinases (TIMPs)라는 억제 단백질에 의해 억제된다. TIMP-2는 21kDa 크기의 포유류 단백질로 대장균에서 과발현 시 다른 많은 포유류 단백질과 마찬가지로 가용성이 낮은 봉입체 형태로 발현된다. TIMP-2 단백질의 접힘에 6개의 이황화결합이 필요하고, 이는 일반적으로 대장균 환경은 적합하지 않다. 본 연구에서는 대장균에서 불가용성으로 발현되는 TIMP-2 유전자를 유전자셔플링 기법의 한 가지인 StEP (staggered extension process)를 변형하고 동시에 $Mn^{2+}$ 농도 변화와 dGTP 불균형을 이용한 무작위 돌연변이 기법을 혼용하여 대장균에서 가용성 TMP-2 재조합 변이체를 생성하고자 하였다. 무작위로 재조합된 TIMP-2 유전자 중에서 가용성으로 발현되는 TIMP-2 유전자를 선별하기 위해서 chloramphenicol acetyltransferase (CAT)-융합 방법을 도입하였다. CAT 유전자가 가용성으로 발현되는 재조합 TIMP-2 유전자에 융합되면 이를 갖는 E. coli는 높은 chloramphenicol 환경에서 생존이 가능하게 된다. 이러한 in virro mutageuesis 기법과 CAT-융합 방법으로 대장균 가용성 TIMP-2 재조합 변이체를 14가지 얻을 수 있었다. 변이체 TIMP-2의 아미노산서열 분석과 구조 분석 결과 주로 소수성 아미노산이 친수성 아미노산으로 전환되었고, MMP와의 결합이 관여하지 않는 C-말단 부위에 돌연변이가 집중되어 있었다. 본 연구에서 개발된 간편하고 새로운 in vitro 재조합 방법과 CAT을 이용한 스크리닝 기법은 다른 많은 대장균 내 불가용성 단백질의 발현에도 사용될 수 있을 것으로 사료된다.