Constructs, transformation and expression analysis
This study aimed at the analysis of the OsGR-RBP4 expression in E. coli M15 cells. To accomplish this, OsGR-RBP4 cloned in pQE30 vector was used. We also used OsGR-RBP4ΔC (truncated version of the Osgr-rbp4 with intact RRM but lacking the glycine rich domain sequences) cloned in pQE30. In addition, we used OsFKBP20 cDNA cloned in pQE30 as control (Figure 1a). As a plasmid control, pQE30 was also transformed in E. coli M15 cells. Four types of recombinant E. coli cells thus analyzed were as per the following constitution: OsGR-RBP4 in M15 cells (OsGR-RBP4/M15), OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15. Selection of the transformed bacterial cells on antibiotic provided first line of evidence that the cells were transformed. Further, expression of the foreign inserts in the E. coli M15 host cells were examined at the transcript level. RNAs isolated from OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 and OsFKBP20/M15 cells were used for cDNA preparation. cDNAs were used for RT-PCR reactions and the products of these reactions were analyzed on 1% agarose gel. As shown in Figure 1b, clear expression was noted for OsGR-RBP4, OsGR-RBP4ΔC and OsFKBP20 RNAs in RT-PCR reaction from the IPTG-induced cells. It is noteworthy that low level expression was also apparent for the IPTG un-induced samples of various transformants (particularly for the OsGR-RBP4ΔC/pQE30 lane). Subsequently, expression of the foreign protein was examined in the E. coli M15 host cells. Total proteins isolated from OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 and OsFKBP20/M15 cells were loaded on 12% SDS-gel. As shown in Figure 1c, the expressed foreign proteins were clearly noted in the IPTG induced samples (shown by arrows). No such protein was detected in E. coli M15 cells transformed with pQE30 plasmid backbone.
Growth analysis of the E. coli M15 cells
Next, growth profiles of OsGR-RBP4/M15, OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15 cells were monitored spectrophotometrically both for the IPTG un-induced and the induced cultures. The overnight grown primary cultures were used to set the secondary cultures beginning with 0.5 O.D. O.D. values were subsequently recorded after every 30 min interval. Growth curves were then plotted for the O.D. values against time intervals. From Figure 2A, it is clear that the growth rates of all four cells types (OsGR-RBP4/M15, OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15) were nearly comparable under IPTG un-induced conditions (shown by blue line color). Strikingly, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells types after IPTG induction failed to grow at all on secondary culturing (shown by pink line color). There was no effect of IPTG addition to pQE30/M15 cell type. While OsFKBP20/M15 cells did grow upon IPTG induction, its growth was slightly less than pQE30/M15 cell type.
To reaffirm the above observations, 5 μl cells from the IPTG un-induced and induced culture were spotted after serial dilutions (10-1-10-3) on LB agar plates with antibiotic selection and allowed to be absorbed by the medium. Similar to the observations made with broth culture, plate assay showed that induction with IPTG has detrimental effect on the viability of OsGR-RBP4/M15 and OsGR-RBP4PΔC/M15 cells. These cells upon culturing on plates showed reduced viability. On the other hand, OsFKBP20/M15 and pQE30/M15 cell types showed no such inhibitory effects (Figure 2B.a).
Next we tested the effect of temperature on bacterial growth. Secondary culture (0.5 O.D. of the overnight grown primary culture used to set the secondary culture) was set up for all the four transformed E. coli M15 cell types. After 3 h, total culture volume was divided into two parts: one part was induced by IPTG and other was left un-induced. After every 30 min interval, cells from the un-induced and induced cultures were serially diluted and spotted on LB agar plates with carbenicillin and kanamycin. Three sets of plates were prepared and one set of each of these was incubated for 16 h at 20°C, 37°C and 45°C in separate incubators. At 20°C (Figure 2B. b) and 37°C (Figure 2B. a), all the four cells types showed comparable growth profile in the sets made without induction by IPTG (Figure 2B. b). In sets prepared with IPTG induced cell types, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells showed lower growth profiles than OsFKBP20/M15 and pQE30/M15 cell types (Figure 2B. c). At both the temperatures of plate incubation, this effect was more pronounced after at least 60 min of IPTG induction. At 45°C (Figure 2B.c), increased lethality to OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells as against OsFKBP20/M15 and pQE30/M15 cells was apparent even at 30 min of IPTG induction. In this stress regime, it was further noted that OsFKBP20M15 and pQE30/M15 cells were able to grow gradually with time following induction by IPTG. However, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells failed to overcome the lethality effects following IPTG induction (Figure 3).
In subsequent experiment, IPTG was washed off from the cultures of all four cell types after inducing it with IPTG for 30 min. The recovered cell cultures were serially diluted and spotted on LB agar plates with antibiotic selection (plates incubated at 37°C for 16 h). The lethality noted for OsGR-RBP4/M15, OsGR-RBP4PΔC/M15 cells was alleviated to a significant extent upon IPTG washing (Figure 3).
Further, growth profiles of E. coli M15 cells were monitored in response to addition of purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins. The respective proteins were partially purified from the corresponding cultures using affinity column chromatography (respective proteins shown by arrows in Figure 4a). Equal amounts of proteins (120 μg) were added directly into the E. coli M15 cultures. It may be noted here that because of the variations in the molecular weights of the purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins, the number of individual protein molecules in 120 μg of protein may vary. After ~8 h of growth, cultures were spotted on LB agar plates with selection (kan). Exogenous addition of the OsGR-RBP4 and OsGR-RBP4PΔC proteins showed a distinct toxic effect on the growth of E. coli M15 cells. Out of these two, OsGR-RBP4PΔC appeared more toxic to cells than OsGR-RBP4. This again may be possible because of higher ability of OsGR-RBP4PΔC protein to enter into E. coli owing to its lower molecular weight. On the other hand, exogenous addition of OsFKBP20 protein didn't result in growth inhibition of bacterial cells (Figure 4b).
Next, we were interested to see if there is any protein in E. coli genome homologous to OsGR-RBP4. OsGR-RBP4 sequence was searched for their homology with E. coli in the NCBI database using BlastP program. We didn't observe any significant homology of OsGR-RBP4 to any E. coli protein in this analysis.