Peptide GVKGDKGNPGWPGAPY, derived from the protein sequence of human collagen type IV, triple-helix domain residues 1263-1277, represents an RGD-independent, adhesion, spreading, and motility-promoting domain in type IV collagen. Its structures and functions were detected at the beginning of 1990's. In order to figure out the relationship between structure and function, this peptide was expressed in repeated form by DNA recombination methods. So that the IV-H1 peptide-coding sequence was decided and the termination region was designed to stop the translation of the peptide-coding gene. The proper sites for restriction endonucleases were located on IV-H1-coding sequence, e.g. EcoRI and Nae1 sites were placed on 5'-terminal and SmaI site was placed on 3'-terminal, for optimizing the ligation into pUC19 and repeating the sequence without changing reading frame. On the termination region, Tyr codon, stop codons, PstI and HindIII sites were located. Two strands of synthesized oligonucleotide, IV-H1 peptide-coding sequence, was phosphorylated, hybridized, and ligated into the linearized pUC19, and named as pUCS1. The annealed termination region was inserted into the linearized pUCS1 with the digestion of SmaI and EcoRI. The resulting plasmid constructed was named as pUCST1. The pUCST1 was confirmed by the PvuII-PstI digestion. The lager size of the pUCS1 fragment digested with AlwNI and SmaI, and the smaller one of the pUCS1 fragment digested with AlwNI and NaeI were eluted. These two strands were ligated. The obtained plasmid was named as pUCS2, which contained 2 times repeated IV-H1 peptide-coding sequence. There was no restriction enzyme site in the linking portion of the two IV-H1 peptide-coding sequences, while NaeI site was kept on the head portion and SmaI site on the tail portion of the pUCS2. In this way, the differently repeated IV-H1 peptide-coding sequences were constructed in pUC19, and designated as pUCSX and pUCSTX (pUCS1, pUCS2, pUCS4, pUCS8, pUCS16, pUCST1, pUCST9, pUCST17, and pUCST33), in which the S stood for the IV-H1 peptide-coding sequences and T for termination sequence following a number X which stood for the times of repetition.
The pMAL-p2, MBP gene fused multicloning plasmid, was chosen as an expression vector. The DNA fragments from pUCST9 and pUCST33 that digested with NaeI and HindIII were inserted into the pMAL-p2 that digested with XmnI and HindIII. The recombinant plasmid was confirmed by the digestion with BglII and PstI. The plasmid, pMAL-p2-T17, which was stored in our lab was confirmed by the digestion with BglII and PstI endonuclease digestion. The fragments were eluted and purified from the agarose gel and religated these two fragments with T4 DNA ligase in order to check the stability of the repeated DNA sequence during gene manipulations. The results indicated that the repeated DNA sequences were stable enough to work on. The DNA sequencing results supported it further. The newly ligated DNA was transformed to E. coli JM109. The successfully ligated pMAL-p2T9, pMAL-p2T17 and pMAL-p2T33 plasmids were primary screened with X-gal plate. The positive colonies were screened further with restriction endonuclease digestion and DNA sequencing with malE primer. These differently repeated peptides IV-H1 were expressed in E coli JM109 as MBP fused form, from which we found MBP-T9 was expressed in a large amount while MBP-T33 was not expressed at all. The expressed repeated peptide IV-H1 was purified with amylose affinity column, quantified with Lowry method and confirmed by western blotting. The cell adhesion function was detected with a modified Chelberg method. The results showed that the repeated peptide adhered to clone 9 cell line in the concentrations of 40 nM. This study indicates that the amount of expressed repeated peptides IV-H1 mainly depended on the times of repetition, and the repeated peptides had the same function of cell adhesion as its monomer did. These results also made clues for studying on proteins' structure and function by combination of theoretical and experimental methods.
인체 콜라젠 IV의 단백질서열에서 유래한 펩타이드인 IV-HI (GVKGDKGNPGWPGAPY)은 콜라젠 IV에서 RGD 비의존성 세포접착, 이동 촉진 기능을 갖는 부위로 알려져 있다. 이 펩타이드의 구조와 기능은 1990년대 초에 알려졌다. 구조와 기능간의 관련상을 알아보기 위해 유전자 재조합기술을 이용하여, 이 펩타이드를 반복형태로 발현하였다. 반복형태의 발현을 위해서 IV-H1 펩타이드의 암호화서열부분과 번역종결부위를 디자인하였다. 반복의 용이함을 위해서 제한효소 절단부위를 암호화서열과 번역종결부위에 인위적으로 도입하였다. 즉 pUC19로의 결찰과 읽는 구조의 변화없는 반복의 편이를 위해 암호화서열의 5′말단에 EcoRI과 NaeI, 3' 말단에 SmaI 절단부위을 도입하였다. 또한 전사종결부위에 티로신 암호화서열과 번역종결서열들, 그리고 PstI과 HindIII 절단부위를 인위적으로 도입하였다. 합성한 상보적인 두 유전자가닥을 인산화와 상호결찰반응 후, pUC19에 삽입시키고 이를 pUCS1이라 명명하였다. 또한 상호결찰된 번역종결부위 유전자를 SmaI과 EcoRI으로 자른 pUCS1과 결찰시키고 pUCST1이라 명명하였다. IV-HI 펩타이드 암호화서열을 두번 반복시키기위해, pUCS1을 AlwNI과 SmaI으로 절단 후 보다 큰 절편과 pUCS1을 AlwNI과 NaeI으로 절단 후 보다 작은 절편을 서로 결찰시킨후, 이를 pUCS2라 명명하였다. 이와 같은 방법으로 pUC19에 다양하게 반복된 IV-HI 펩타이드 암호화서열을 얻었다. 이들 각각을 pUCSX와 pUCSTX (pUCS1, pUCS2, pUCS4, pUCS8, pUCS16, pUCST1, pUCST9, pUCST17 그리고 pUCST33)라 이름 지었다. 여기서 S는 IV-HI 펩타이드 암호화서열을, T는 전사종결서열의 존재유무를 그리고 숫자 X는 IV-HI 펩타이드 암호화서열의 반복횟수를 의미한다.
반복된 IV-HI 펩타이드 암호화서열의 발현을 위해서 pMAL-p2라는 접합발현벡터를 선택하였다. NaeI과 HindIII로 절단한 pUCST9과 pUCST33의 유전자를 XmnI과 HindIII로 자른 pMAL-p2에 삽입하였다. 이 만들어진 플라스미드를 BglI과 PstI으로 확인하였고, 유전자 재조합 과정에서 이들 반복된 유전자가 충분한 안정성을 가짐을 확인하였다. 성공적으로 결찰된 pMAL-p2T9, pMAL-p2T17 그리고 pMAL-p2T33을 일차적으로 X-gal 배지에서 확인하였고, 일차 확인된 클론들을 제한효소 절단과 유전자 서열 분석으로 확인하였다. 이렇게 얻은 다양하게 반복된 IV-HI 펩타이드를 대장균에서 maltose-binding protein과 융합된 형태로 발현에 성공하였다. 발현 결과 9번 반복된 IV-HI 펩타이드 경우 상당량이 발현된 반면 33번 반복된 IV-HI 펩타이드경우에는 거의 발현되지 않았다. 발현된 반복 IV-HI 펩타이드를 아밀로우스 친화성 컬럼 (amylose affinity column)으로 분리하여 western blotting으로 확인하였다. 정제한 반복 IV-HI 펩타이드의 세포접착능을 변형된 Chelberg 방법으로 확인하였다. 확인 결과, 반복 펩타이드가 40 nM 의 농도에서 clone 9 세포에 부착하였다. 이상의 연구를 통해서 반복된 펩타이드의 발현에 그 펩타이드의 반복 횟수가 중요한 요인이며, 반복 펩타이드가 원래의 단일체 (monomer)와 같이 세포접착 기능을 유지하고 있다는 사실을 알 수 있었다. 이러한 결과는 또한 이론과 실제 실험간의 조합을 통해 단백질의 구조와 기능간의 상관성을 알아내는데 중요한 단서를 제공해 줄 수 있으리라 사료된다.
