A method to use Chlorella to express a recombinant heterologous protein that can be recovered from the extracellular medium has been developed. Plasmids are constructed with an extracellular secretion signal sequence inserted between a promoter region and a gene for human growth hormone (hGH). The plasmids also contain a Kanr region which confers resistance to the antibiotic G418. Protoplasts are prepared by enzymatic treatment, and the plasmid is introduced by incubation of the protoplasts with polyethylene glycol and dimethyl sulfoxide. Cells are then grown in the presence of G418, and the medium is collected from 6 days after transfection. hGH is measured by immunoassay, and values for expressed hGH of about 200-600 ng/ml are obtained.

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ABSTRACT.Transcriptional regulation of the gene for the myeloid calcium binding protein, MRP14, was investigated in humanmonocytic leukemia cell lines. The MRP14gene was not expressed in monoblastic ML-1 cells, promonocytic U-937 cells, or promyelocytic HL-60 cells. On the other hand, the gene was expressed in monocytic THP-1 cells and in the HL-60 cells treated with 1,25-dihydroxyvitamin D3 (VD3). The level of MRP14in VD3-treated HL-60 cells was two-fold higher than that in THP-1 cells. Amongseveral known transcription factor binding motifs, nuclear protein(s) of VD3-treated HL-60 cells and THP-1 cells bound to the CCAAT/enhancerbinding protein (C/EBP)-binding motif that was located in the upstream region of the MRP14gene (-81), as evidenced by the competitive gel mobility-shift assay. An antibody for C/EBPa supershifted the nucleoprotein complex in THP-1 cells but not in the VD3-treated HL-60 cells, whereas an antibody for C/EBP^blocked the formation of the complex with the nuclear factor of the HL-60 cells but not with that of THP-1 cells. An anti-C/EBPd antibody had no effect on the complex in either cell. Thus, it was concluded that C/EBPa and -fi were able to bind to the C/EBP motif, and that C/EBPa bound to the motif in THP-1 cells and C/EBP^bound to that in the VD3-treated HL-60 cells. Furthermore, to examine the transcriptional activity of the C/EBP motif, we transfected several constructed luciferase reporter DNAsinto HL-60 cells and THP-1 cells. The luciferase activity of the C/EBP motif in HL-60 cells was increased by VD3treatment. The C/EBP motif in the MRP14gene was confirmed to function as a regulatory region in VD3-treated HL-60 cells and THP-1 cells by the assay. Since C/EBP/9 was also detected in VD3-untreated HL-60 cells by immunoblotting, VD3 activated C/EBP/9 to bind to the motif, probably through post-translational modification.Myeloid calcium binding proteins, MRP8and MRP14 (25, 35), also designated cystic fibrosis antigen (5, 12), the leukocyte LI molecule (4), the 60B8 antigen (34, 55), p8/pl4 (17), calgranulin A/B (22), and calprotectin (51, 66), are members of the S100 protein family, whose genes are knownto be localized in the cluster on human chromosome Iq21 (46). Therefore, it has been proposed that MRP8and MRP14be called S100A8 and S100A9, respectively (46).

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