MOLECULAR-CLONING AND EXPRESSION OF MEGAKARYOCYTE POTENTIATING FACTOR CDNA
MOLECULAR-CLONING AND EXPRESSION OF MEGAKARYOCYTE POTENTIATING FACTOR CDNA
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DOI：
10.1074/jbc.270.37.21984
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发表时间：
1995-09-15
影响因子：
4.8
通讯作者：
YAMAGUCHI, N
中科院分区：
文献类型：
--
作者：
KOJIMA, T;OHEDA, M;YAMAGUCHI, N
人巨核细胞增强因子（hMPF）先前已从人胰腺癌细胞HPC-Y 5的培养上清液中纯化（Yamaguchi，N.，服部，K.，Oh-eda，M.，小岛，T.，北伊迈，和Ochi，N.（1994）J.Biol.Chem.269，805-808）。我们现在已经从HPC-Y 5 cDNA文库中分离hMPF cDNA，使用聚合酶链反应和噬斑杂交方法。hMPF cDNA编码由622个氨基酸组成的多肽，包括33个氨基酸的信号肽，并且具有68 kDa的推导分子量，尽管HPC-Y 5细胞分泌33-kDa形式的hMPF。人MPF与其它先前描述的序列没有显示出任何显著的同源性。该cDNA在COS-7和中国仓鼠卵巢（CHO）细胞中表达，并在其培养上清中检测到巨核细胞增强活性。COS-7细胞仅分泌33-kDa的重组hMPF，而在CHO细胞的培养基中检测到另外的30-kDa形式。从CHO细胞中纯化的33-kDa rhMPF显示出巨核细胞增强活性，但纯化的30-kDa rhMPF没有。33-kDa和30-kDa形式的hMPF之间的结构和活性差异归因于33-kDa形式的C-末端25个氨基酸残基的存在。
The human megakaryocyte potentiating factor (hMPF) has been previously purified from a culture supernatant of human pancreatic cancer cells HPC-Y5 (Yamaguchi, N., Hattori, K., Oh-eda, M., Kojima, T., Imai, N., and Ochi, N. (1994) J. Biol. Chem. 269, 805-808). We have now isolated hMPF cDNA from a HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The hMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular mass of 68 kDa, although HPC-Y5 cells secrete a 33-kDa form of hMPF. Human MPF does not show any significant homology with other previously described sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and megakaryocyte potentiating activity was detected in their culture supernatant. The COS-7 cells secreted only a 33-kDa recombinant hMPF, whereas an additional 30-kDa form was detected in the culture medium of CHO cells. The 33-kDa rhMPF purified from CHO cells showed megakaryocyte potentiating activity, but not the purified 30-kDa rhMPF. The difference in structure and activity between the 33- and 30-kDa forms of hMPF was ascribed to the existence in the 33-kDa form of the C-terminal 25 amino acid residues.