Molecular cloning, expression and characterization of serine protease from the olive flounder (Paralichthys olivaceus)
- Alternative Title
- 넙치로부터의 serine protease 의 분자생물학적 클로닝, 발현, 특성분석
- Abstract
- 일반적으로 serine proteases의 촉매활성은 활성부위에 존재하는 3개의 아미노산(Ser, His and Asp)나타나게 되며 진핵생물과 원핵생물에서 발견되어진다. Serine proteases는 구조를 기준으로 chymotrypsin-like 와 subtilisin-like로 구별할수 있다. Chymotrypsin-like serine proteases 의 경우 두개의 beta-barrel domains 을 가지고 있으며, 기질에 따라서 trypsin-like, chymotrypsin-like, elastase-like로 나눠진다. Serine protease는 척추와 무척추 동물에서 세포의 생리적,병리적 신호를 증폭시키는 역활을 한다고 알려져 있다. 포유동물에서는 혈전용해와 미생물감염과 관계가 있는 것으로 보고되고있다.
본 실험에서는 넙치에서 elastase-like Serine protease를 클로닝하였다.넙치로부터 동정된 serine proteases는 269개의 아미노산을 암호화하는 807bp의 open reading frame 을 가진다.
RT-PCR을 수행한 결과, 넙치의 간에서 발현되는 것을 확인할 수 있었다.
promature 형태의 유전자를 대장균에서 과발현시켰다. 재조합 단백질은 pH7.5및 40℃의 배양환경에서 최고 활성을 나타냈으며, 기질은 Z-Phe-Arg-AMC을 이용하여 측정하였다.
We have cloned a cDNA encoding a Elastase-like serine protease (ELSp ) from the olive flounder, Paralichthys olivaceus. The ELSp gene was determined to consist of the 807 bp nucleotide sequence which encodes for a 269-amino acid polypeptide. The tissue-specific pattern of ELSp was examined by RT-PCR. The results of RT-PCR analysis revealed liver, kidney and spleen the entirety of the flounder tissues, and the expression of the ELSrP gene was also examined in brain, gill, kidney, spleen, and muscle at 0,1,3,6, and 24H after induction of an artificial bacterial infection (lipopolysaccharide, LPS). The cDNA encoding enzyme of ELSp was expressed in Escherichia coli using the pET32a vector systems. The activity of ELSp was detected by gelatin zymography and cleaving synthetic fluorogenic Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of serine protease. The optimal pH for the protease activity was 7.5
- Author(s)
- 한진욱
- Issued Date
- 2013
- Awarded Date
- 2013. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- URI
- https://repository.pknu.ac.kr:8443/handle/2021.oak/24679
http://pknu.dcollection.net/jsp/common/DcLoOrgPer.jsp?sItemId=000001966057
- Alternative Author(s)
- Han, Jin woo
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 생물공학과
- Advisor
- 이형호
- Table Of Contents
- LIST OF TABLE ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••Ⅱ
LIST OF FIGURE ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••Ⅱ
KOREAN ABSTRACT •••••••••••••••••••••••••••••••••••••••••••••••••••••••••Ⅳ
1. Introduction ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••1
2. Materials and Methods
2.1. cDNA synthesis from olive flounder and rapid amplification of cDNA ends (RACE) •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••3
2.2. Sequence and phylogenetic analysis••••••••••••••••••••••••••••••••••••••••••••••4
2.3 LPS-injected espression study by a quantitative method•••••••••••••••••••••••••5
2.4. Expression studies by RT-PCR & quantitative real-time PCR••••••••••••••••••6
2.5. Expression and purification of recombinant pro- PoESp in E. coli•••••••••••••8
2.6. SDS-PAGE, western blotting and zymography••••••••••••••••••••••••••••••••••9
2.7. Enzyme activity assays••••••••••••••••••••••••••••••••••••••••••••••••••••••••••10
2.8. Effect of enzyme inhibitors, metal ions and detergents••••••••••••••••••••••••11
3. Results & Discussion
3.1 Cloning and sequence analysis of Paralichthys olivaceus elastase-like serine protease cDNA••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••13
3.2. Tissue distribution of PoESp ••••••••••••••••••••••••••••••••••••••••••••••••••••13
3.3. Enzymatic characterization of recombinant Pro-PoESp••••••••••••••••••••••••14
REFERENCES•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••31
ABSTRACT ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••34
ACKNOWLEDGEMENT•••••••••••••••••••••••••••••••••••••••••••••••••••••••36
- Degree
- Master
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