Rubisco expressions

Transcriptional inhibitors could be used simultaneously. Rifampicin blocks chloroplast and mitocondrian RNA synthesis [23, 24], while tagetitoxin is a very specific inhibitor of chloroplast RNA polymerase [25]. Treatment with these antibiotics does not inhibit Rubisco SSU synthesis since the promoter is part of the nuclear genome, while the cytosolic ribosomes are not affected by streptomycin. Therefore SSU promoters can be used to drive transgene expression and facilitate the accumulation of recombinant proteins. Expressed proteins are targeted to a suitable cellular compartment, such as the cytoplasm, apoplastic space or chloroplast, depending on the nature of the protein. 

As discussed above, Rubisco levels have been reduced by expressing antisense RNA in transgenic tobacco plants [26]. Plants expressing antisense rbcS RNA showed reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced levels of LSU and SSU proteins. 

We have isolated Rubisco small subunit promoters from several plant species and tested their strength with gusA and ALB (human serum albumin) transgenes in sprouts. The highest level of expression in Brassica napus sprouts has been obtained... 

The best results are obtained when 100 mg I. 1 streptomycin is added 48-50 h after the initiation of germination. With streptomycin treatment, 100-400% increases in recombinant protein expression have been obtained. The accumulation of both Rubisco subunits is prevented (Figure 3.7). The specific activity of GUS increases 2.5-fold when streptomycin is used (Figure 3.8). 

Fig. 3.6 Human serum albumin (HSA) was produced during the sprouting of transgenic rape-seeds. A light-inducible Rubisco promoter was used to control transgene expression. The highest yield was obtained with light induction after three days of continuous darkness. Sprouting was carried out in an airlift fermentor at room temperature. The sprouting medium was water.

Fig. 3.8 Transgenic rapeseeds expressing the gusA reporter gene were germinated in an airlift tank with streptomycin added to the medium (100 mg L 1). Streptomycin was added 0, 38, 42 or 50 h after germination. When streptomycin is added after 50 h, a 2.5-fold increase in GUS activity can be seen. This indicates the importance of correct timing when streptomycin is added to inhibit endogenous Rubisco gene expression.

DeRocher, E.J. Bohnert, H.J. (1991). Post-transcriptional regulation of rubisco small subunit gene expression during environmental stress and development. Plant Physiology 96S, 83. 

It is assumed that the reaction catalyzed by RubisCO is a rate-limiting step in reactions of photosynthetic carbon fixation expressed as follows in the lump ... 

Robinson and colleagues (2003) explored the properties of rubisco from the bacterial endosym-biont of the giant tube worm Riftia pachyptila. Rubisco, from any source, catalyzes the reaction of either C02 (Fig. 20-7) or 02 (Fig. 20-20) with ribulose 1,5-bisphosphate. In general, rubisco reacts more readily with C02 than 02. The degree of selectivity (IT) can be expressed as... 

To investigate the regulation of the expression of two RubisCO, studies are in progress utilizing both photoautotrophic conditions and photoheterotrophic conditions. 

When the cDNA encoding human CA was introduced into Synechococcus sp. PCC7942, CA was expressed in the cytoplasm and the transformant showed a high CO2 requiring phenotype, possibly due to a rapid conversion of cytoplasmic bicarbonate into CO2, followed by a CO2 leakage from the cells to the medium[2]. This showed the importance of CA localization in the cells. In order to evaluate the effect of increased CO2 supply to RuBisCO on CO2 fixation, we constructed transformed Synechococcus sp. PCC7942 which overexpressed CA at the site of RuBisCO. In this report, we examined the effect of CA over-expression on CO2 fixation rate. 

By immunological electron microscopy, CA protein was found in carboxysomes of transformants, where most of RuBisCO protein was also found, but CA protein was not found in cytoplasm, showing that the site specific expression of CA in the cells [6]. 

Figure 2 Chaperonin- and Mg-ATP-dependent in vitro reconstitution of active dimeric Rubisco from denatured Rubisco. a) Rubisco activity, expressed as a percentage of activity observed with an equal quantity of native Rubisco. b) Western blot after non-denaturing PAGE of the reaction mixtures used in a and probed with antibody raised against Rubisco. c) as in b, but probed with antibody raised against cpn60. d) Summary of the reaction conditions. Reprinted by permission from NATURE vol. 342 pp. 884-889. Copyright (C) 1989 Macmillan Magazines Ltd.

Roberts RB, Abelson PH, Cowie DB, Bolton ET, Britten RJ (1955) Studies of Biosynthesis in Escherichia coli. Carnegie Irrstitution of Washington Publ 607, Washingtorr, DC Robinson JJ, Cavanaugh CM (1995) Expression of form 1 and form 11 Rubisco in chemoautotrophic symbioses Implicatiorrs for the interpretation of stable carbon isotope values. Lirtmol Oceanogr 40 1496-1502... 

---