Yeong-Man Hong

Molecular cloning and sequencing of the epidermal cell differentiation inhibitor gene from Staphylococcus aureus

We recently purified to homogeneity a protein inhibiting differentiation of cultured keratinocytes from extracellular products of Staphylococcus aureus, and named it epidermal cell differentiation inhibitor (EDIN). In the present study, we isolated and sequenced the structural gene coding for EDIN from Staphylococcus aureus E-1 using oligonucleotide probes on the basis of the partial amino acid sequence of the purified EDIN. DNA sequencing of the cloned DNA revealed an open reading frame encoding 247 amino acids as a precursor of EDIN, which included an NH2-terminal signal sequence of 35 amino acid residues. Processing of this precursor produces a mature EDIN protein composed of 212 amino acids with a calculated Mr of 23,782. The EDIN shared 35% amino acid homology with the ADP-ribosyltransferase C3 of Clostridium botulinum. These results with biological properties of EDIN described previously indicate that EDIN is a novel protein.

Purification and characterization of recombinant human interleukin-1β produced in Escherichia coli

Summary Recombinant human interleukin-1 β (rIL-1 β ) produced in Escherichia coli was purified to homogeneity by a combination of mass ion exchange column chromatography, ion exchange and gel filtration high performance liquid chromatography. The purified rIL-1 β had a molecular weight of 18 kD on SDS-polyacrylamide gel electrophoresis and an isoelectric point of 6.9 on analytical isoelectric focusing. These values were almost same as those of natural interleukin-1 β . The amino acid composition and amino acid sequence of the amino terminal region were consistent with those deduced from the cDNA sequence. In addition, the primary structure was confirmed by peptide mapping with lysyl-endopeptidase on reverse phase HPLC. Besides rIL-1 β with amino terminal Ala, two molecular species, [Met 0 ]rIL-1 β and [desAla 1 ]rIL-1 β , were also obtained. Biological and physicochemical properties of the three species of rIL-1 β were compared.

Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins

By means of reverse‐phase HPLC, 2 different proteins were obtained from apparently purified pig cosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre‐proGMBP2 with 3 domains; signal peptide, acidic pro‐portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein: but no such similarity existed in the pro‐portion. Although the molecular weights determined by SDS‐PAGE of guinea pig and human MBPs were 11 000 and 9 300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the bisic domains of mature proteins, were well conserved between guinea pig and human MBPs.

Macrophage colony-stimulating factor is produced by human T lymphoblastoid cell line, CEM-ON: Identification by amino-terminal amino acid sequence analysis

A subclone, designated CEM-ON, derived from an azaguanine-resistant human leukemic T cell line, CEM-AG(R), constitutively secretes a colony-stimulating factor (CSF) which stimulates the production of macrophages from murine bone marrow progenitor cells. This CSF has been purified from serum-free conditioned medium. Highly purified CEM-ON CSF with a specific activity of 4.7 X 10(7) units/mg protein was obtained. Amino-terminal sequence analysis showed that the first 27 amino acids were identical to the amino-terminal sequence of the M-CSF (CSF-1) based on the cDNAs for human M-CSF. On SDS-PAGE analysis, CEM-ON CSF had an apparent molecular weight of 33,000-43,000; following reduction with 2-mercaptoethanol, this migrated as a 20,000-24,000 subunit, suggesting a homodimer structure. These results show that a human T cell line, CEM-ON, secretes M-CSF into its medium.

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 μg/ml both in the presence and in the absence of extracellular Ca2+. After reverse‐phase high‐performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pl values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPx was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 × 103 clones, a positive clone, which encoded a full length of pre‐proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre‐proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.