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Crystal Structure of CRISPR-associated Protein Cas2 from Xanthomonas albilineans
Xanthomonas albilineans에서 유래된 CRISPR-associated Protein Cas2의 결정 구조

DC Field Value Language
dc.contributor.advisor배의영-
dc.contributor.author정유진-
dc.date.accessioned2017-07-14T06:47:08Z-
dc.date.available2019-11-28T06:35:53Z-
dc.date.issued2016-08-
dc.identifier.other000000136699-
dc.identifier.urihttps://hdl.handle.net/10371/125960-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2016. 8. 배의영.-
dc.description.abstractClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 시스템은 외부로부터 유입된 파지 (phage) 또는 플라스미드 (plasmid) 등에 대항하는 고세균과 세균에서 발견되는 방어기작이다. Cas (CRISPR associated) 단백질들은 면역 반응 기관으로서 RNA를 기반으로 작용된다. 이는 후천적으로 얻은 면역력의 세대 계승이 가능하다. 모든 CRISPR 시스템은 Cas1과 Cas2를 가진다. Cas2는 보편적으로 보존되어 있는 단백질이며 새로운 스페이서 (spacers)의 유입에 관여한다. 단백질 결정화를 통해 몇 몇의 Cas2 단백질의 구조가 규명되었다 그 예로서, Bacillus halodurans (BhCas2) Desulfovibrio vulgaris (DvCas2), 그리고 Streptococcus pyogenes (SpCas2) 이 존재한다. 모든 Cas2 단백질은 N 말단에 ferredoxin fold (βαββαβ)와 C 말단에 짧은 310 helix와 다섯 번째 β-strand를 가진다. 본 논문에서 이용된 Xanthomonas albilineans는는 Subtype Ⅰ-C의 CRISPR를 가지고 있으며 Cas1, Cas2, Cas3, Cas4, Cas5d, Cas7, Cas8의 Cas 단백질로 이루어져 있다. 본 논문에서는 Xanthomonas albilineans에서 유래된 Cas2 (XaCas2) 단백질의 발현, 분리 및 정제, 결정화 한 후 X-ray 결정 구보 분석법을 이용하여 단백질의 3차 구조를 규명 하였다. 정제된 단백질로 결정화를 시도 했을 때 20℃와 4℃에서 결정을 얻을 수 있었고 그 구조적인 특성은 전형적인 Cas2의 특징을 가지고 있었으며, 온도가 다르다 하더라도 그 모양이 서로 같았다. 20℃에서 자란 결정은 1.65 Å으로, 4℃에서 자란 결정은 1.75Å으로 데이터를 모을 수 있었다. XaCas2는 수소결합 (hydrogen bonds)과 염다리 (salt bridges) 로 다이머 (dimer)를 유지하고 있으며 다른 동족체 (homologues)와 시퀀스 (sequence)와 3차원적 구조를 비교했을 때 Cas2 단백질의 구조는 기질 (substrate)이 접근할 때 변화 할 것으로 예측된다.-
dc.description.abstractClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system that provides RNA-based adaptive immunity to archaea and bacteria is a defense system against extrachromosomal genomic elements. And their variable CRISPR-associated (Cas) proteins are known as immune effectors which are composed of distinct subtypes. There are three steps in CRISPR-Cas system-
dc.description.abstract(1) Acquisition (2) Processing of crisprRNA and (3) Interference. All CRISPR systems have Cas1 and Cas2 proteins. cas1 and cas2 genes are core cas genes in all CRISPR-Cas subtypes. The cas2 gene from Xanthomonas albilineans GPE PC73 genomic DNA (Gene ID: 8702143) was amplified by polymerase chain reaction for generation of a C-terminal His tag and it was cloned into pET21a. The Xacas2 gene was expressed in Escherichia coli BL21 (DE3). The XaCas2 protein was purified by two steps and I could get pure XaCas2 protein to make protein crystals. The protein crystals were made in a condition that contains 0.17 M ammonium acetate, 0.085 M sodium acetate pH 4.6, 25.5% (w/v) polyethylene glycol 4000 and 15% (v/v) glycerol. The conditions of protein crystals were optimized for making big and angulate protein crystals. The protein crystals of XaCas2 were made at two different temperatures, 20 ℃ and 4℃. The structures of XaCas2 were solved with molecular replacement. Both structures of XaCas2 consist of N-terminal ferredoxin fold and C-terminal segment which contains a short 310-helix and a fifth β-strand. And there was no difference between two structural features. However, conformational differentiations were found in C-terminal end between A and B protomers within each crystal structure. The fifth β-strand crosses the four stranded β-sheets on the other protomer, making five stranded antiparallel β-sheets. The swapping of C-terminal segment stabilize the dimerization. The dimerization is supported by hydrogen bonds and salt bridges in β-sheets. Comparing with other Cas2 homologues, XaCas2 likely to have double strand DNases activity.-
dc.description.tableofcontentsIntroduction 1

Materials and Methods 7
Cloning of Xacas2 gene 7
Protein expression and purification of XaCas2 7
Crystallization of XaCas2 8
X-ray data collection 9
Model building and refinement 9

Results 12
Expression and purification of XaCas2 12
Crystallization and X-ray analysis of XaCas2 12
Crystal structures of XaCas2 22
Dimer interface of XaCas2 29
Structural comparison with other Cas2 homologues 29

Discussion 47

References 51

Abstract in Korean 54
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dc.formatapplication/pdf-
dc.format.extent2428214 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectCas2-
dc.subjectClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-
dc.subjectCRISPR associated (Cas) proteins-
dc.subjectXanthomonas albilineans GPE PC73-
dc.subjectx-ray crystallography-
dc.subject.ddc630-
dc.titleCrystal Structure of CRISPR-associated Protein Cas2 from Xanthomonas albilineans-
dc.title.alternativeXanthomonas albilineans에서 유래된 CRISPR-associated Protein Cas2의 결정 구조-
dc.typeThesis-
dc.description.degreeMaster-
dc.citation.pages55-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2016-08-
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Master's Degree_농생명공학부)
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