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><tbody><tr><td>pET22b (+)</td><td>Novagen</td><td>69744</td><td>T7 expression vectors with resistance to ampicillin&amp;nbsp;</td></tr><tr><td>BL21(DE3)pLysS&amp;nbsp;</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>1 M 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&amp;trade; 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 ...

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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

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 ...

연구

생체공학

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

전체 피부 결함 모델은 생체의 혈관 신생을 평가하기<em&gt; 생체</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

기질 자체 히드로 생체 외에서 그리고 Vivo에서 응용 프로그램에 대 한 조립으로 인간의 심장 조직 처리

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

발생학

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

압축 된 콜라겐에 다진 조직 : 단일 무대 재건 복구를위한 세포 함유 Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

개선 2D 및 3D 스킨<em&gt; 체외</em거대 분자의 크라우 딩 사용&gt; 모델

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

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, ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

캡슐화 및 3d에서 셀의 Immunostaining에 대 한 엘라 스 틴 같은 단백질 Hydrogels의 생산

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

희생 빈 섬유 막 세포 배양을 통해 기질 섬유의 생산

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

자가 조립 혈관 인체 피부 등가물의 생성

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

설계 및 인공 세포 외 기질의 건설 (aECM) 단백질에서

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