Stable production of an analog of human tissue plasminogen activator from cultured Drosophila cells.

We have studied the expression of an analog of human tissue plasminogen activator, FK2P, in Drosophila Schneider 2 cells. A number of promoters were tested, including the Drosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE), Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1 alpha promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 micrograms FK2P/10(6) cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 microgram FK2P/10(6) cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominantly as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.

Secretion of active truncated CD4 into Escherichia coli periplasm.

A truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli. The signal sequence from the E. coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter. The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120. Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane. A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E. coli can be optimized by manipulating the host secretion apparatus.

A simple Escherichia coli system for monitoring HIV protease activity: analysis of two temperature-sensitive protease mutants.

A simple Escherichia coli system has been developed for the detection of human immunodeficiency virus (HIV) protease activity. In this system, the protease sequence is placed downstream of the HIV gag polypeptide in an operon arrangement. Upon expression of the operon, gag serves as the substrate for the protease; the level of protease activity can be determined by measurement of the cleavage product of gag in cell extracts by Western immunoblotting. This system is useful in both detection of protease mutations generated by mutagenesis and in testing substrate specificity of the protease by mutagenesis of the gag sequence. Using this system, we have observed that modification of the N-terminus of HIV protease renders the enzyme temperature sensitive; the temperature sensitivity is made more pronounced by the conserved change of valine to isoleucine at residue eleven.

Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E. coli.

We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E. coli. The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E. coli using a pBR322-derived vector. However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product. Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA. There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation. To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out. The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation. Removal of this stem-loop results in a 100-fold increase in BSt expression. However, the expression level is still low, amounting to only 0.5-1% of total cell protein. High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons. These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation.