The coiled-coil neck domain of human pulmonary surfactant protein D drives trimerization and stabilization of thioredoxin, a heterologous non-collagenous protein.
The coiled-coil neck domain of pulmonary surfactant protein D (SP-D) is required for trimeric association and the subsequent assembly of functional dodecamers of SP-D. It is also necessary and sufficient for trimerization of a heterologous collagen sequence. To investigate whether it is capable of driving trimerization of heterologous non-collagenous proteins, we expressed and purified a fusion of a heterologous non-collagenous sequence (thioredoxin) to the coiled-coil neck domain of human SP-D here. While western blot analysis detected a small population of stable trimers of the fusion protein, chemical cross-linking and SEC-HPLC indicated that the fusion protein was predominantly a trimer. In contrast, purified thioredoxin without the fusion was found only as monomers and dimers. We also measured the thermal stabilities (with circular dichroism) and degradation rates of these two proteins. Our data showed that the fusion protein had a melting temperature that was 13 K higher than that of thioredoxin and a longer degradation half life than thioredoxin. Our findings indicate that the coiled-coil neck domain of SP-D enables the trimerization and stabilization of the heterologous non-collagenous thioredoxin. It may provide new clues for further study on the application of this human original coiled-coil domain in protein engineering to construct trimeric functional fusion proteins.
Li
Ping
P
College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
Zhou
Jian Ying
JY
Zhou
Yin Yan
YY
Qian
Chao Dong
CD
Li
Ou
O
Min
Hang
H
Wu
Xue Chang
XC
D000595:Amino Acid Sequence; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D037663:Pulmonary Surfactant-Associated Protein D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D037663:Pulmonary Surfactant-Associated Protein D; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins