Studies on expression and cell adhesion function of repeated peptide IV-H1

Abstract

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, pUCS1...