저자는 교육부의 AcRF 계층 1 (RG41)에서 자금을 인정하고 난양 기술 대학에서 부여를 시작하고 싶습니다. 저와 Tjin은 난양 기술 대학, 싱가포르에서 연구 학생 장학금 (RSS)에 의해 지원된다.

aECM 단백질 재조합 플라스미드 인코딩 1. 복제 <ol><li> 기능 도메인 (예, 세포 결합 도메인과 엘라스틴 같은 반복)의 아미노산 서열을 디자인. 기능 영역의 단부의 측면에 디자인 제한 부위는 서브 클로닝 소프트웨어 명령에 따른 무료 소프트웨어를 사용하여 (예 http://biologylabs.utah.edu/jorgensen/wayned/ape/)을 용이하게한다. 여기서, 의도 사이트로 소화를 한정하기 위해 기능 도메인에 존재하지 않는 유일한 제한 효소 부위를 선택한다. 다중 클로닝 부위 호스트 벡터 (MCS)에 표시 제한 부위를 선택 (예를 들어, pET22b (+))의 서브 클로닝. </li><li> 소프트웨어 명령어에 따라 웹 사이트를 이용하여 자유 염기 서열에 아미노산 서열을 해석 역방향. (예 http://www.bioinformatics.org/sms2/rev_trans.html). 확인 코돈은 E.에 최적화되어 있습니다 대장균 호스트. </li><li> 관심의 유전자는 purchas 수 있습니다ED 상업적로서 단일 가닥 올리고 뉴클레오티드는 DNA 및 어닐링을 수행 (올리고 &lt;100 염기쌍 (BP)이있는 경우, 즉) (단계 1.4 참조) 비용을 감소시킨다. 그렇지 않으면, 100 염기쌍보다 큰 유전자들은 상업적인 회사에서 구입할 수있다. </li><li> DNA 올리고 뉴클레오티드의 어닐링 <ol><li> 원하는 유전자 서열을 얻기 위해 DNA 올리고머 어닐링. 1 μg의 / μL의 최종 농도 (여과 2- 아미노 -2- (히드 록시 메틸) 프로판 -1,3- 디올, pH가 8.0, 10 mM 트리스) DNA 올리고머 버퍼 DNA 올리고 뉴클레오티드를 녹인다. </li><li> 총 40 ㎕의 혼합물을 달성하기 위해 DNA 어닐링 완충액 32 μL (10 mM의 트리스, 100 mM의 NaCl 내지 100nm의 MgCl 2)에 각 올리고머 4 μl를 추가. </li><li> 핫 플레이트를 사용하여 물을 비이커를 삶아 5 분, 95 ° C의 혼합물을 담그지. 비커를 제거하고 점차적으로 스티로폼 상자 O / N에서 설정 전체를 냉각. 올리고머는 어닐링 및 소화 할 준비가되어있다. </li></ol></리&gt; <li> 어닐링 올리고머 (즉, 삽입 라한다) 및 호스트 벡터 별도로 (즉, pET22b (+)를) 소화 해당 제한 효소를 추가합니다. 37 ℃에서 3-4 시간 동안 다음 레시피 (1-2 μg의 DNA 각각을 제한 효소 2㎕의, 총 50 ㎕의 혼합물을 10 배 제한 효소 완충액, 및 물을 최대 5 μL)을 사용. 참고 : pET22b (+) 플라스미드 벡터는 암피실린 항생제 내성과 C- 말단에 6 배 - 그의 태그가 포함되어 있습니다. pET22b (+)에 존재하는 6 배-그의 태그는 7 단계에서 웨스턴 블로 팅을 통해 표적 단백질을 식별하기 위해 그의 - 태그 항체를 사용하여 우리를 할 수 있습니다. </li><li> 각 소화 혼합물에 6 배 로딩 염료를 추가합니다. 별도로 100 V. 시각화 UV 광 조명과 1.2 %의 DNA 아가 로즈 겔에서 1 시간 동안 UV 형광 DNA 얼룩을 함유하는 1.2 % DNA 아가 로스 젤에서 DNA 사다리 포함 소화 혼합물을 실행. </li><li> 상업 젤 정화 키트를 사용하여 소화 DNA 제품을 추출하기 위해 젤을 조각입니다. 엘 루트E 50-100 NG / μL의 최소 DNA 농도를 달성하기위한 최소한의 열을 사용하여 볼륨. </li><li> 소화 된 DNA 인서트를 결찰하여 관심 순차적 유전자를 결합 (즉, DNA의 어닐링에 의해 얻어지는 엘라스틴 반복 또는 세포 결합 도메인)하기 배합하여 T4 리가 제를 사용하여 플라스미드 벡터로 (벡터의 2 μL, T4 리가 제 1 μL를, T4 내고 버퍼, X μL 삽입의 1.5 μL, 총 15 μL 혼합물 10.5-X μL 물). 2 시간 동안 실온에서 결찰 혼합물을 품어. 참고 : 삽입하는 벡터의 몰 농도는 결찰 효율을 최적화하기 위해 변화되어야한다. 조리법에있는 X 설명 삽입물의 볼륨 단계 1.7에서 용출 DNA 농도에 따라 달라집니다. </li><li> 해동 E. 대장균 DH5α 화학적으로 유능한 세포 (또는 복제 주) 얼음에. 따뜻한는 2xYT 아가 플레이트 암피실린 (25 μg의 / ㎖)를 함유하는 (표 1)에 37 ° C. </li><li> 사용하여 세포를 형질열 충격 : <ol><li> 나누어지는 깨끗하고 사전 냉장 마이크로 원심 분리기 튜브에 유능한 세포의 50 μL. 피펫 5 μL 세포로 결찰 혼합물 (100 페이지 NG 100 사이), 피펫 팅 부드럽게 위아래로 혼합합니다. 20 분 동안 얼음에 혼합물을 남겨주세요. </li><li> 2 분 동안 42 ° C의 물을 용기에 세포 혼합액을 함유하는 마이크로 원심 분리 튜브를 담그고 2 분 동안 얼음에 돌려 보냈다. 세포에 열 손상을 최소화하기 위해 시간을 침지 시간. </li><li> 마이크로 원심 분리기 튜브에 SOC 미디어 (표 1)의 500 μl를 추가하고 1 시간 동안 진탕 37 ° C에서 품어. </li><li> 암피실린 (25 μg의 / ㎖)를 포함, 실온으로 미리 예열 된 함유하는 2 × YT 한천 플레이트에 세포 / 결찰 혼합물 (50) 500 μl를 확산하고 판을 37 주에서 거꾸로 품어 / N (12 ~ 16 시간) . </li></ol></li><li> 다음날, 깨끗한 피펫 팁을 사용하여 한천 플레이트에서의 DNA 식민지를 선택합니다. (표 2YT 배지 5ml에 콜로니를 성장1)을 포함 암피실린 (25 μg의 / ㎖) O / N) 진동 (225 rpm으로 37 ° CO / N (12 ~ 16 시간)에서. </li><li> 다음 날, 제조 업체의 프로토콜에 따라 고른 각 식민지에 대한 DNA 플라스미드를 추출하는 플라스미드 분리 키트를 사용합니다. 물 50 μL와 DNA를 용출시켰다. </li><li> 37 ° C에서 2 시간 동안 배양 (5 μL의 DNA 각각을 제한 효소 0.2 μL, 총 10 ㎕의 혼합물을 1 μL 10 배 제한 효소 완충액 및 상부까지 물) : 시험은 다음의 레서피를 이용하여 제한 효소로 소화 수행 가능성이 단계 1.6에서 1.2 %의 DNA 아가로 오스 겔에 삽입하고 실행을 포함 식민지 화면. 참고 : 소화시, 성공적인 결찰해야 두 밴드, 벡터 및 각각의 분자량에 해당 삽입 (그림 1)에 대한 하나의 각각의 결과. </li><li> 앞으로 T7 프로모터를 사용하여 DNA 염기 서열에 대한 가능성이 식민지를 보내기 및 상업 시퀀서에 프라이머를 역. </li></ol><ol><li> 시퀀싱 결과로부터 성공적으로 결찰 된 콜로니를 선택하고 E.로 형질 전환 DNA 플라스미드를 사용 대장균 발현 호스트. </li><li> 해동 E. 대장균 발현 균주 얼음 (BL21 (DE3) pLysS를). 한편, RT를 암피실린 (25 μg의 / ㎖) 및 클로람페니콜 (34 μg의 / ㎖)를 함유하는 한천 판 따뜻한. 주 : 박테리아에 존재 pLysS를 플라스미드는 BL21 (DE3) pLysS 균주는 클로람페니콜 내성 유전자를 포함 균주. pLysS를 플라스미드는 구조적으로 aECM 단백질의 새는 표현을 제한하는 표현 T7 리프레 유전자가 포함되어 있습니다. 클로람페니콜 pLysS 균주는 배양 중에 포함되어 박테리아 세포에 대해 선택하는 것이 필요하다. </li><li> 단계를 반복 1.10 E. 변형을 구하는 인공 단백질을 발현 할 준비가 대장균 세포. 파라 필름에 한천 플레이트를 감싸고 4 거꾸로 저장 6; 최대 1 개월 C. </li></ol> aECM 단백질의 3 세균 식 <ol><li> 도 2를 피펫 팁으로 변환 된 한천 플레이트에서 식민지를 선택하고 멸균 좋아요 국물 (TB) 미디어 시험관에 암피실린과 클로람페니콜 항생제를 모두 포함 (표 1) 10 ㎖에 접종을 참조하십시오. 225 rpm으로 진탕 37 ° CO / N (12 ~ 16 시간)에서이 스타터 문화를 품어. </li><li> 이전 3 L 삼각 플라스크에 같은 항생제로 보충 한 L 신선한 멸균 결핵 매체에 스타터 문화 10 ㎖. 2 ~ 3 시간 동안 225 rpm으로 진탕 37 ° C에서 문화를 품어과 문화 (OD 600)의 광학 밀도를 관찰 읽기 빈 큐벳에 문화 1 ㎖를 피펫 팅에 의해 0.6-0.8에 도달했습니다. 이전 SDS-PAGE 특성화 유도에 문화 1 ㎖를 저장합니다. <ol><li> 세포 배양액의 OD (600)를 측정하기 위해, 큐벳에서 TB 매체의 1 바이알을 제조빈 측정. 별도로, 새로운 빈 베트로 문화 플라스크에서 배양 1 ㎖를 전송합니다. 600 nm의 흡광도에서의 제어에 대해 빈 분광 광도계를 사용하여 배양 물의 광학 밀도를 측정한다. </li><li> 2 분 동안 12,000 XG에 1.5 ml의 microcentrifuge 관, 원심 분리기로 문화 샘플의 1 ml에 전송하여 (이후의 SDS-PAGE 분석을위한) 샘플을 저장하고 상층 액을 가만히 따르다. </li></ol></li><li> 최종 농도 1 mM로 이소 프로필 β-D-1- 티오 갈 락토 피 라노 시드 (IPTG)를 배양을 유도하고 다른 4 시간 동안 225 rpm으로 진탕하면서 37 ℃에서 배양한다. SDS-PAGE 특성화를위한 4 시간에서 유도의 끝에서 문화 1 ㎖를 저장합니다. </li><li> 4 ℃에서 30 분 동안 12,000 XG에서 1 리터의 원심 분리기 병 원심 분리기에 문화를 전송하여 세포를 수확. G / ㎖ 0.5 (pH는 8.0, 1 M 트리스, 0.01 M EDTA, 0.1 M의 NaCl), 상층 액을 버린다 TEN 버퍼에 세포 펠렛을 Resuspend 무게. </li></ol> 세균 배양 4. 용해 <ol><li> -80 ° CO / N에서 다시 정지 세포 배양을 동결. 세포를 용해시키기 위해 RT 또는 얼음 수욕 냉동 세포 배양액을 해동. 해동 동안 10 μg의 / ㎖ 디옥시리보 뉴 클레아 제 I (DNase의 I), 10 μg의 / ㎖의 리보 뉴 클레아 제 A (의 RNase I) 및 50 μg의 / ㎖의 페닐 메틸 설 포닐 플루오 라이드 (PMSF)를 첨가하고, 천천히 교반하면서 용액을 균질화한다. </li><li> 모든 세포를 재현 탁하고, 해동 후에 물 12 단백질의 용해도를 증가하여 pH 9.0 용액을 조정한다. 동질적인 일관성을 달성하기 위해 얼음에 교반 현명한 6 N NaOH를 드롭을 추가합니다. 2 mm 직경 플랫 팁, 5 초 펄스를 사용하여 얼음에 20 분 동안 초음파 중단에 의해를 Lyse. </li><li> 4 ℃에서 30 분 동안 12,000 XG에 세포 용액을 원심 분리기. 나중에 정화 4 ℃에서 깨끗하고 빈 병 및 저장소에 뜨는을 전송합니다. </li><li> 한편, -80 ° C에서 TEN 완충액 재 동결 다시 세포 펠렛을 재현 탁. 에세 번에 완전한 세포 용해를 반복 동결 / 해동 및 초음파 프로세스입니다. SDS-PAGE 특성 20 μL 세포 용 해물을 저장합니다. </li></ol> 역 전환 자전거를 사용 aECM 단백질의 정제 (5) <ol><li> 단계 4.3에서 세포 용 해물을 부씩과 엘라스틴 계 단백질 (21)의 정제를 위해 사용되는 것과 유사한 ITC를 사용 aECM 단백질을 정제로 진행. 사이클 4 ° C에있는 다른 온도로 수행되며, 37 ° C ( &quot;콜드&quot;라한다)는 각각 사이클 ( &quot;웜&quot;라 함). </li><li> 4 ℃에서 2 시간 동안 40,000 XG에서 세포 50ml의 원심 분리기 병에 해물, 원심 분리를 분할합니다. 세포 펠렛의 모양은 암갈색하고 콧물 보일 것입니다. </li><li> 펠렛에서 깨끗한 분리를 얻기 위해 피펫으로 상층 액을 제거합니다. 깨끗한 병 원심 상등액을 수집하고 (3 M에서 최대) 1 M의 최종 농도의 NaCl를 추가한다. 따뜻한225 rpm으로 진탕 2 시간 동안 37 ° C의 솔루션입니다. 참고 : 염화나트륨의 추가가 aECM 원인 집계 엘라스틴 성분의 전환을 트리거합니다. 상층 액을 탁 켜집니다. 단백질 농도가 충분히 높은 경우, 흰색 거품 단백질은 원심 분리 병의 측면에서 볼 수있다. </li><li> 37 ℃에서 2 시간 동안 40,000 XG에 단계 5.3에서 뜨는을 원심 분리기. 뜨는을 가만히 따르다. 금속 주걱을 사용하여 펠렛을 분쇄 및 자기 교반 막대 및 플레이트를 이용하여 4 ℃에서 격렬히 교반 O / N와 빙냉 멸균 증류수 펠릿 비트 (50 ㎎ / ㎖)에 재현 탁. 펠렛을 완전히 용해한다. </li><li> 반복 단백질의 높은 순도를 얻기 위해 5.2 다른 3-5 회 5.4 단계를 반복합니다. </li><li> 정제의 최종 사이클 후, 4 ℃에서 증류수에 대하여 투석을하여 단백질 용액을 탈염. 물 매 4의 변화에​​ 2-3 일 동안은 물에 대한 단백질 용액을 투석시간 또는 O / N 8 시간. SDS-PAGE에 대한 정제 된 단백질의 20 μl를 저장하고 더 사용할 때까지 -80 ° C에서 정제 된 단백질과 상점의 나머지 부분을 동결 건조. </li></ol> SDS-PAGE 전기 영동을 사용하여 단백질의 aECM 6. 특성화 <ol><li> (분자량이 큰 경우, 더 나은 분리를 위해 8 % SDS-PAGE 겔을 사용) 12 % SDS-PAGE 겔을 준비한다. 각 시료에 2 × SDS 로딩 버퍼를 추가 가열 샘플을 10 분 동안 100 ° C에서 실행 샘플을 겔에서 1 시간 동안 100 V 또는 aECM 단백질의 분자량에 상응하는 단백질 사다리의 중간에 도달 할 때까지 젤. </li><li> 설정 SDS-PAGE에서 젤을 검색하고 페트리 접시에 1 시간 동안 젤 잠수함하는 볼륨 쿠마에게 브릴리언트 블루 얼룩 (표 2)를 추가합니다. 로커에 5 분 동안 탈색 용액 (표 2)에 린스 젤. 탈색 솔루션을 변경하고 T의 배경까지 흔들와 젤을 destain 계속그는 젤은 분명해진다. 단백질 밴드가 명확하게 볼 수 있어야합니다. </li><li> 표적 단백질의 분자량을 결정하기 위해 단백질 래더 대해 표적 단백질의 위치를​​ 비교한다. </li><li> 상기 표적 단백질의 존재를 확인하기 위해, 단계 7과 웨스턴 블롯을 수행한다. </li></ol> 웨스턴 블로 팅을 사용하여 aECM 단백질 7. 특성 <ol><li> 아니오 염색 부 (6)에서와 같이 12 % SDS-PAGE 겔상에서 샘플을 실행. </li><li> SDS-PAGE 젤보다 약간 큰 크기로 니트로 셀룰로오스 막 잘라. 동일한 크기의 여과지 4 조각을 잘라. </li><li> 웨스턴 전송 버퍼 여과지 젖은 원피스 (20 % V / V를 메탄올, 25 mM 트리스, 190 mM의 글리신, pH를 8.3), 여과지의 다른 조각 뒤에 여과지 위에 SDS-PAGE 겔을 올려 다음, 니트로 셀룰로오스 막 및 필터 종이의 마지막 다른 두 조각. 확인 전체 셋업 충분한 서부 전송 버퍼로 침수입니다. 참고 : 단백질이 접촉시 막에 결합 수로 젤 막은이 시점에서 접촉해서는 안된다. </li><li> 니트로 셀룰로오스 막이어서 웨스턴 반건식 이송 유닛으로 두 여과지를 배치하여 니트로 셀룰로오스 막 상에 단백질을 전송하고, 신중 내에 멤브레인에 SDS-PAGE 겔을 배치, 마지막에 마지막 두 젖은 여과지를 배치 SDS-PAGE 젤. 45mA, 30 분에서 서쪽 전송을 실행합니다. 전압보다 높은 30 V. 경우 버퍼를 추가 참고 : 바람직 시도와 멤브레인의 SDS-PAGE 젤을 배치하고 단백질이 접촉시 막에 결합시킬 수 있으므로 막에 불필요하게 젤을 이동하지 마십시오. </li><li> 검색하고 RT에서 2 시간 동안 (여과지로 여과 PBS pH를 7.4에서 5 % 무 지방 우유) 차단 용액으로 니트로 셀룰로오스 멤브레인을 차단. 버퍼를 차단 폐기와 린스 PBS를 추가합니다. 참고 : 새 장갑으로 변경 원치 않는 보호 자전거와 막 오염을 방지하기 위해인. </li><li> 1 PBS에 희석 차 안티 그의 항체를 품어 : 1,000 실온에서 1 시간 동안 락을 함께. 1000 항체의 1 μL (스톡 농도 1 ㎎ / ㎖)을 추가 희석 비율 (1)과, 예를 들어, 전체 막 잠길 수있는 부피 혼합물을 제조 한 용액 전체 혼합물을 999 ㎕의 PBS로. </li><li> (0.1 % 트윈 20과 함께 PBS) PBST 5ml를 가진 막을 씻어. </li><li> 1 PBS에 희석와 말 무 퍼 옥시 다제 (HRP)에 접합 된 이차 항체와 함께 품어 : 5,000 실온에서 1 시간 동안 락을 함께. 5000 항체의 1 μL (스톡 농도 1 ㎎ / ㎖)을 추가 희석 비율 (1)과, 예를 들어, 전체 막 잠길 수있는 부피 혼합물을 제조 5 ml의 전체 혼합물에 대한 4999 μL의 PBS로. </li><li> PBST 5 mL로 막을 세척하고 두 번 반복합니다. </li><li> 멤브레인을 커버하고 사용하는 기판의 지침에 따라 부화 볼륨 막에 화학 발광 기판을 적용합니다. 참고 : 단백질 검출 기판의 감도가 다를 수 있습니다, 우리는 신호를 증가시키지 않습니다 항체 농도를 증가시 매우 낮은 신호를 최대 감도와 기판을 권장합니다. </li><li> CCD 카메라 기반 이미 저를 사용하여 화학 발광 신호를 캡처. 신호가 약한 및 비특이적 인 경우 일차 및 이차 항체의 희석 비율을 조정한다. 배경 신호가 하이 인 경우 차단 용액에 이상 인큐베이션. </li></ol>

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E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

재조합 단백질 공학은 상향식 접근을 사용하여 신규 한 단백질 물질을 생성하는 다용도 기술이다. 단백질 계 물질은 관심있는 용도에 따라, 복수의 기능을 갖도록 설계 맞추어 질 수있다. 인해 복제 및 단백질 발현을 증가시키는 기술의 발전에, 그것을 재현하고 확장 가능한 방식으로 인공 단백질의 다양성을 생성하기 위해 상대적으로 간단한 (그리고 비용 효율)이되었다. 엘라스틴 유사 도메인?...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</td>
			<td>Invitrogen</td>
			<td>C6060-03</td>
			<td>additional antibiotics - chloramphenicol</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>1M stock solution with autoclaved water, make fresh prior to induction.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>plasmid isolation kit</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Affymetrix</td>
			<td>23660</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean&#8482; gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-gold strain</td>
			<td>Agilent Technologies</td>
			<td>200315</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

엘라스틴 형상의 반복을 포함하는 융합 단백질을 설계에서, 전체 콘텐츠 엘라스틴, 융합 단백질 (18)의 충분한 부분을 유지하는 것이 중요하다. 이 융합 단백질 작 제물이 정제를 위해 ITC를 사용하기 위해, 그것의 엘라스틴과 같은 특성을 보유하고 있는지 확인하는 것이다. aECM 단백질 설계 및이 절에서 설명하는 순서는 특히 Tjin 등. (14)에 의해 직장에서 촬영되었다. ?...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

설계 및 인공 세포 외 기질의 건설 (aECM) 단백질에서<em&gt; 대장균</em&gt; 피부 조직 공학

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

design-construction-artificial-extracellular-matrix-aecm-proteins

Research

JoVE Journal

Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

8.8K 조회수.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

비디오: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

라만 분광학을 사용하여 세포와 세포외 기질의 비 접촉, 레이블이없는 모니터링

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

연구

생체공학

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

조직 공학을위한 이축 기계 로딩 생물 반응기의 설계

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

조직 공학 : 계층화 된 세포 시트의 전달을위한 다세포 3D 비계 건설

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

상피 세포 다시 채우기 및 신장 조직 개발을위한 설치류 세포 외 기질 비계의 준비

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

세포 외 매트릭스 내에서 임베디드 공학 3 차원 상피 조직

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

브릴 루앙 분광학에 대한 세포 외 기질 단백질 섬유의 제조

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

조직 특이 적 세포 배양 및 전달 플랫폼과 같은 세포 외 기질 유래 거품과 마이크로 담체의 제조

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

날조는 신장 피 질 세포 외 매트릭스 파생 하이드로 겔

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...