Characteristics of metalloprotease and its inhibitor protein purified from recombinant serratia재조합 serratia 균주에서 정제한 metalloprotease와 그 억제단백질의 특성

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Metalloptease(SMP) from Serratia marcescens is a zinc-containing protease that is secreted from S. marcescens. Inhibitor(SmaPI) of metalloprotease from S.marcescens, SMP, is a protein which reduces or blocks the activity of SMP with high specificity. Using ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephacryl S-200 chromatography, SMP was purified to homogeneity from the culture medium of the recombinant Serratia marcescens. Also, Secreted SmaPI was purified to homogeneity via a series of purification steps including ammonium sulfate precipitation, heat treatment, ultrafiltration, Mono-Q chromatography, and Superose 12HR 10/30 chromatography. The pIs of SmaPI and SMP were estimated to be about 10 and 8.2, respectively. Both two proteins were basic proteins. Nα-Benzoyl-DL-Arg-ρ-nitroanilide was used as an artificial substrate, and various kinetic parameters of SMP obtained as follows; $K_m= 2.4mM$, $K_{cat}=0.13sec^{-1}$, $K_{cat}/K_m=0.054 sec^{-1}mM^{-1}$. Analysis of the kinetic data for the SMP digestion of Nα-benzoyl-DL-Arg-ρ-nitroanilide revealed that SmaPI was a competitive inhibitor with an inhibitory constant($K_i$) of 0.2nM. Nucleotide sequencing of smapi gene suggests the existence an N-terminal signal peptide and a second ATG codon. Proteolysis of SmaPI by SMP at various conditions was not observed even in one week reaction time.
Advisors
Byun, Si-MyungChung, Jae-Hoon변시명정재훈
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
1995
Identifier
98684/325007 / 000933100
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생명과학과, 1995.2, [ vi, 54 p. ]

Keywords

단백질 분리

URI
http://hdl.handle.net/10203/28434
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=98684&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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