OBJECTIVESTo express human testis Lipocalin-type prostaglandin D synthase in Pichia Pastoris for further research on biological function and clinical applications.

METHODSHuman testis L-PGDS gene coding region was amplified from plasmid pGEX-2T/htL-PGDS by PCR with a deletion of the signal peptide sequence. The DNA fragment was inserted into pPIC9 to construct yeast expression plasmid followed by transformation of the yeast GS115 strain with electroporation. The recombinant his-tag protein was induced to express by methanol.

RESULTSThe sequence of the amplified DNA fragment was identical to that of human testis L-PGDS previously reported. The recombinant protein was found with a molecular mass of 27,000 on SDS-PAGE, which was identical to that of native L-PGDS.

CONCLUSIONSSecretory expression of human L-PGDS was obtained in Pichia Pastoris.

Cloning, Molecular ; Gene Expression ; Humans ; Intramolecular Oxidoreductases ; biosynthesis ; genetics ; Lipocalins ; Male ; Pichia ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; secretion

OBJECTIVE: To express and purify the extra cellular full-length human apolipoprotein M(ApoM). METHODS: The ApoM gene fragment was amplified from the human liver cDNA library by PCR. The resulting product was cloned into pGEXT vector and sequenced. Then the confirmed canstatin cDNA was cloned into plasmid E.coli JM109 and then transformed into E.coli DL21(DE3) where it was induced to express protein by IPTG. RESULTS: The ApoM gene was cloned by PCR and a 560 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoM gene in GenBank. Then ApoM cDNA gene fragment was induced by IPTG, and a 24 kD recombinant ApoM protein was tested on SDS-PAGE. CONCLUSION Human ApoM gene is successfully cloned and its recombinant proteins are expressed.
Apolipoproteins ; biosynthesis ; genetics ; isolation & purification ; Apolipoproteins M ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Lipocalins ; Molecular Sequence Data ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

SIP24/24p3 is a secreted murine acute phase protein which has been speculated to play an anti-inflammatory role in vivo. Recently SIP24/24p3 has been found to be able to specifically induce apoptosis in leukocytes. By using (35)S metabolic labeling method, we studied the regulation of SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines IL-6 and TNF-alpha in cultured Balb/c 3T3 and BNL cells. The following results were observed: (1) dexamethasone induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells, the induction was more significant in BNL cells; (2) dexamethasone and IL-6 synergistically induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells; (3) in Balb/c 3T3 cells dexamethasone and TNF-alpha acted synergistically to induce the expression of SIP24/24p3, whereas in BNL cells dexamethasone and TNF-alpha induced the expression of SIP24/24p3 in an additive manner; (4) dexamethasone and IL-6/TNF-alpha acted synergistically in Balb/c 3T3 cells and additively in BNL cells to induce the expression of SIP24/24p3. The inducibility of SIP24/24p3 by multiple factors will help to explain its highly specific expression in vivo. The difference in the expression patterns of SIP24/24p3 in different cell types is also suggestive to its expression and regulation in hepatic and extrahepatic tissues. Finally, the fact that SIP24/24p3 protein can be induced by both pro-inflammatory as well as anti-inflammatory factors is indicative of the important role of SIP24/24p3 in the entire acute phase response process.
Acute-Phase Proteins ; biosynthesis ; genetics ; Animals ; BALB 3T3 Cells ; Carrier Proteins ; biosynthesis ; genetics ; Cytokines ; pharmacology ; Dexamethasone ; pharmacology ; Drug Synergism ; Gene Expression Regulation ; Interleukin-6 ; pharmacology ; Lipocalin-2 ; Lipocalins ; Mice ; Mice, Inbred BALB C ; Oncogene Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; pharmacology

In a previous search for the differentially expressed genes in keratinocyte differentiation, we identified neutrophil gelatinase-associated lipocalin (NGAL) as a calcium- induced gene. In this study, we further verified the expression of NGAL in cultured keratinocytes as well as in several skin diseases. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and ELISA clearly showed that NGAL expression was markedly increased in calcium-induced keratinocyte differentiation in vitro. However, in our previous report, NGAL expression was not detected in normal skin tissue except for hair follicle by in situ hybridization and immunohistochemistry, indicating the difference of cell status between in vitro and in vitro conditions. Interestingly, NGAL expression was highly increased in psoriasis-like inflammatory disorders (lichen planus and pityriasis rubura pilaris) and skin cancers (keratoacanthoma and squamous cell carcinoma), implying that NGAL may be related with the epidermal hyperplasia. Collectively, these results reveal the potential importance of NGAL in the maintenance of skin homeostasis.
Acute-Phase Proteins/*biosynthesis ; Calcium/*metabolism ; Cell Differentiation ; Culture Media ; Culture Media, Conditioned ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Homeostasis ; Humans ; Keratinocytes/enzymology ; Lipocalins/*biosynthesis ; Models, Biological ; Proto-Oncogene Proteins/*biosynthesis ; Psoriasis/enzymology ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/*metabolism ; Skin Neoplasms/enzymology