日本囊对虾Kunitz型蛋白酶抑制剂在毕赤酵母中的表达纯化及活性分析

SKPI(shrimp Kunitz-type protease inhibitor)是日本囊对虾(Marsupenaeus japonicus)体内的一个小分子多肽, 含有一个Kunitz型结构域, 属于丝氨酸蛋白酶抑制剂。目前已知丝氨酸蛋白酶抑制剂在节肢动物免疫系统中起着非常重要的作用, 为了了解SKPI在对虾天然免疫系统中的作用, 首先对其进行了重组表达。从日本囊对虾肝胰腺中扩增skpi的cDNA片段, 插入改造后的pPIC9K酵母表达载体, 获得的重组质粒转化至毕赤酵母GS115进行表达。由于改造的pPIC9K载体加入了6-His标签, 因此利用Ni?Sepharose?High?Performance对SKPI进行了高效纯化。初步的活性研究表明, 重组表达的SKPI能特异性地抑制胰蛋白酶的水解活性。     

Primary structure of kunitz-type trypsin inhibitor-2a (pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a,pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a,pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2,pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1,pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.     

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein.All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.     

Matriptase Expression and Zymogen Activation in Human Pilosebaceous Unit

Studies of human genetic disorders and mouse models reveal the important roles of matriptase in hair growth. Here, we investigate matriptase expression and zymogen activation in hair follicles. We show: 1) layer-dependent distribution patterns, with much higher matriptase expression in cells of the outer root sheath and matrix cells of the hair bulb than in cells of the inner root sheath; 2) cycle-dependent expression patterns, with matriptase expressed in the anagen and catagen phases of the hair lifecycle, but not in the telogen phase; 3) reduced expression of the matriptase inhibitor, HAI-1, in the catagen phase, suggesting increased proteolytic activity in this phase; and 4) definitive matriptase zymogen activation patterns, with the highest matriptase activation observed in matrix cells and outer root sheath cells in the isthmus/bulge region. In sebaceous glands, matriptase is highly expressed in basal and ductal cells, with much lower expression in the differentiated, lipid-filled cells of the interior. We also show that matriptase potently activates hepatocyte growth factor (HGF) in vitro, and that the HGF receptor, c-Met, is co-expressed in those cells that express activated matriptase. Our observations suggest that the matriptase-HGF-c-MET pathway has the potential to be engaged, primarily in proliferative cells rather than terminally differentiated epithelial cells of the human pilosebaceous unit.     

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.     

Detection of immunologically related Kunitz and Bowman-Birk proteinase inhibitors expressed during potato tuber development

Antiserum against a potato Kunitz-type proteinase inhibitor (PKPI) expressed in Escherichia coli was produced. In immunoblotting assays of proteins from potato tubers cultured in vitro, three proteins reacted to the antiserum, two of 20 kDa and one of 10 kDa. Their N-termini were sequenced. While the 20 kDa proteins showed 59 and 90% identity to PKPI, the 10 kDa one had 65% identity to soybean C-II proteinase inhibitor. Characterization of the temporal expression of these proteins showed that both could be detected from 10 days after induction of tuberization (DAI) in vitro, but the times when maximum amounts of PKPI and 10 kDa protein could be detected were different, corresponding to 22 and 32 DAI, respectively. The amounts of these proteins decreased in the following stages, and no positive reaction of the antiserum with mature tuber proteins could be found. The 20 kDa proteins were also detected in early stages of development of potato tubers grown in the field, indicating that these proteins are expressed during normal tuber development, and differ from the PKPIs reported previously.