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Vascular endothilial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties. VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PlGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PlGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions in vivo.

Two different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (bFGF) using Escherichia coli TGl as host organism. The bFGF structural gene was cloned into two vectors differing only with respect to the promoter, which was either the bacteriophage λ P R P L - or the E. coli lac -promoter. The resulting expression systems were studied in high-cell density cultures. Cells were grown in a fed-batch procedure with a predetermined exponential feeding rate based on mass balances and kinetic equations to ensure constant specific growth rates. Prior to induction, cells were grown at 30°C. Product formation was induced by either a temperature shift from 30 to 42°C or by the addition of isopropyl-β- d -thiogalactopyranoside (IPTG). Under comparable culture conditions—induction of bFGF expression at 41 to 45 g l −1 dry cell weight and the addition of feed medium with identical rates—bFGF accumulated to 4.9 and 1.1 g l −1 using the temperature- and IPTG-inducible expression systems, respectively. The final biomass concentrations obtained using temperature- and IPTG-inducible expression systems were 61 and 135 g l −1 dry cell weight, whereas the specific product concentrations were 80 and 8.1 mg bFGF g −1 dry cell weight, respectively. When a temperature shift was used for product induction, 30% of bFGF was recovered as inclusion bodies in the insoluble cell fraction. IPTG-dependent induction yielded exclusively soluble bFGF.

Using antibodies raised against the purifiedi protein, the expression of the chromosomaluncI gene was demonstrated. Thei protein was identified as a component of the cytoplasmic membrane and shown to be present in preparations of F0 or F1F0. The protein is not associated with the F1 moiety.

Differential expression of the genes in the Escherichia coli atp (unc) operon is achieved via control of the translational initiation, translational coupling and mRNA stability of the respective genes. The atpIB region of the polycistronic mRNA is less stable than the remaining seven genes. We have investigated the functional half-lives of the atp genes in reconstructed versions of the operon. In order to be able to do this reliably, we have readdressed the interpretation of the complex functional inactivation data obtained by means of transcriptional inhibition using rifampicin. Our results indicate the usable information to be gleaned from this commonly applied technique, while identifying the potential errors in their quantitative interpretation. We estimate that the functional half-life of atpB is slightly over one-half that of atpE and the other atp genes, while atpI is at least two times less stable than atpB. The instability of the atpI mRNA was also demonstrated by its rapid fragmentation. Relocation of atpIB to a position in the promoter-distal region of the operon between atpG and atpD did not change the inactivation rate of atpB. However, it did destabilize the atpG mRNA. Examination of the physical degradation of atpI mRNA shows particularly rapid cleavage in this gene, thus explaining the destabilization effect. The atpIB segment is therefore an autonomously unstable region that can act as a destabilizing element for upstream-located genes in a polycistronic environment.

A new strategy is described for the production of recombinant heteromultimeric proteins using Escherichia coli as host. A recombinant operon was constructed containing modified cDNA sequences encoding the mature A and B chains of human platelet-derived growth factor (PDGF). The relative expression rates of the PDGF genes were varied over a range equivalent to A:B ratios from 0.8 to 3.7 by means of translational regulation. This was achieved using two different translational initiation sequences (TIS) upstream from the respective coding regions, one derived from the E. coli atpE translational initiation region, and the other containing a sequence with extended complementarity to the 3' end of the 16S rRNA. The generation of mature PDGF A and B chains in different relative amounts in E. coli provided the basis for developing a novel procedure for the production of the biologically active PDGF heterodimer AB in large quantities. The general strategy is applicable to the preparation of a wide range of heteromultimeric complexes. Moreover, the described PDGF operon constitutes a compact and versatile model system for studies of the posttranscriptional regulation of gene expression.