DELETION OF A LARGE DOMAIN IN RECOMBINANT HUMAN PROCOLLAGEN-II DOES NOT ALTER THE THERMAL-STABILITY OF THE TRIPLE-HELIX

A construct of the human gene for procollagen II (COL2A1) was prepared with an internal deletion of 5 kilobases that removed 12 exons coding for 291 amino acids from near the NH2 terminus of the triple helix. T he construct was then used to transfect stably a human tumor cell line (HT-1080), and clones secreting internally deleted proalpha1(II) chai n of procollagen II were isolated. The protein was purified, and the t hermal stability of the triple-helical domain was assayed by brief pro tease digestion. The thermal stability of the internally deleted prote in was the same as that of intact collagen II even though the triple h elix was 39% shorter. Additionally, the thermal stability of the colla genase A fragment was the same as that of the collagenase A fragment o f normal collagen II even though it was 38% shorter. Analysis of the r esults suggested that the thermal stabilities of large fragments of co llagen II depended primarily on their contents of -Gly-Pro-Hyp-triplet s corrected for length.