Solubility studies, rational amino acid replacements and structural analyses of streptomyces jumonjinensis isopenicillin n synthase 2

Chapter 4.2 Results Investigation of factors that may influence expression of sjIPNS in E. coli The ability to rapidly produce high levels of recombinant proteins is a critical link between the discovery of new genes and the identification of targets for drug development. Presently, the expression and purification of protein targets greatly lag behind the enormous new gene sequences generated. Understandably, the bottleneck is due to the absence of a universal expression system that can efficiently express vast number of proteins with different characteristics. And often time, the recombinant protein may end up being insoluble when it is highly expressed, just like in the case of sjIPNS. As such, sjIPNS becomes a suitable model protein for us to study ways to overcome insolubility problem during protein expression. For a start, this section focuses on modification of various expression conditions to possibly achieve high-level production of soluble sjIPNS. These factors include the use of an alternative host strain, fusion partners and wider range of induction temperatures. To ensure that the expression results were comparable, non-recombinant control cultures and recombinant E. coli cultures were grown to O.D.600 of ~1.2-1.3, induced with 1mM IPTG and incubated for 15 hours. With the exception of experiments for studying effects of induction temperature, all other experiments were carried out at 37°C and 25°C, the two reference temperatures used for grouping IPNS isozymes based on their solubility at these temperatures. 4.2.1 Host strain E. coli harbors proteases that are liable to degrade foreign proteins and cause aggregate formation (Gottesman, 1990; Enfors, 1992). Consequently, different strains of E. coli possessing unique sets of proteases can influence the foreign protein expressed to different extent (Kenealy et al., 1987; Obukowicz et al., 1992). To circumvent this limitation, a common approach is to use E. coli strains deficient in certain proteases to allow accumulation of recombinant proteins at high rates by decreasing the chances of protease degradation during Chapter Results expression. The E. coli strain, BL21(DE3), used in the expression of different IPNS isozymes in Section 4.1.4 does not produce the lon and OmpT proteases. Although large amounts of soluble enzymes of bacterial scIPNS, sfIPNS, nIPNS and sIIPNS and fungal cIPNS have been produced in E. coli BL21(DE3) at the respective optimal temperatures (Fig. 4.9), sjIPNS protein overexpressed in this strain remains insoluble. The wide array of commercially available E. coli deficient strains for protein expression has allowed us to use an alternative strain, E. coli BLStar, to examine whether this choice of host strain can increase the production of soluble sjIPNS. In addition to lon and OmpT mutations, E. coli BLStar also carries mutation in gene coding for RNaseE which is responsible for degradation of mRNAs. The lack of RNaseE would reduce the susceptibility of heterologous mRNAs to degradation, resulting in more heterologous mRNAs being available for protein translation and hence has been proposed to enhance protein production. The genotype of the strain is described in Table 3.2. To facilitate this study, recombinant construct pET-SJ was transformed into E. coli BLStar. The recombinant and non-recombinant E. coli BLStar were expressed at 37°C and 25°C and the respective protein fractions were analyzed by SDS-PAGE (Fig. 4.11a). No overexpressed protein band was observed in both the soluble (S) and insoluble (I) fractions of the non-recombinant E. coli BLStar control culture and hence only the soluble fraction was shown in lane marked C1 of Fig. 4.11aI. Majority of the 37kDa sjIPNS overexpressed band was associated with the insoluble fractions of E. coli BLStar (Fig. 4.11aI). The expression results of sjIPNS in E. coli BL21(DE3) have already been discussed in Section 4.1.4. Here, the results are presented alongside with the E. coli BLStar expression results for comparison (Fig. 4.11aII). The percentages of insoluble sjIPNS expressed in E. coli BLStar were measured by densitometric scanning (Fig. 4.11a). A slightly higher level of insoluble sjIPNS was expressed in E. coli BL21(DE3) compared to E. coli BLStar, however, the difference is only marginal. In both hosts, only ~2-5% of soluble sjIPNS were detected at 37°C and 25°C. Chapter Fig. 4.11 Results Investigation of factors that may influence expression of soluble sjIPNS in E. coli. The table below indicates the respective recombinant E. coli cultures used for expression studies of sjIPNS (a) in different E. coli host strains; (b) as NusA and GST fusion proteins and (c) at wider range of induction temperatures. The first lane (marked M) in all gels shows the protein standards of various molecular sizes (kDa). The second lane marked C1 in (a)I shows the soluble fraction of E. coli BLStar. The second lane marked C2 in all gels except (a)I shows the soluble protein fraction of E. coli BL21(DE3). The respective soluble and insoluble protein fractions were marked S and I accordingly. The arrows indicate the positions of sjIPNS expressed. The percentages of sjIPNS expressed in various protein fractions were measured using densitometric scanning and the values obtained were plotted in the charts shown. Factors Recombinant E. coli cultures Induction temperature (a) Host strain pET-SJ/ BL21Star pET-SJ/ BL21(DE3) 37°C, 25°C (b) Fusion protein pGST-SJ/ BL21(DE3) pNUSA-SJ/ BL21(DE3) 37°C, 25°C (c) Induction temperature pET-SJ/BL21(DE3) 37-15°C (a) Host strain I. E. coli BLStar II. E. coli BL21(DE3) 37°C kDa 100 75 M C1 S I 25°C S 37°C kDa I M C2 S 25°C I S I 100 75 50 50 37 37kDa 25 37 37kDa 25 % of sjIPNS expressed in different E. coli hosts at 37°C and 25°C 37°C 35 25°C 37°C 25°C % of sjIPNS expression 30 25 20 15 10 S I S E. coli BLStar I S I S I E. coli BL21(DE3) 105 Chapter Results Fig. 4.11 (ctd.) (b) Fusion protein II. Nus-A I. NusA-sjIPNS 37°C kDa 250 150 100 75 M C1 S 37°C 25°C I S I kDa 92 kDa 50 37 37 25 25 III. GST-sjIPNS 25°C I S I 55kDa IV. GST 37°C M S 150 100 75 50 kDa M C1 C1 S 25°C I S 115 83.0 63 kDa 49.4 25°C 37°C kDa 150 100 75 I M C1 S I S I 50 37 34.6 29.0 % of recombinant protein expression 35 25 37°C 37°C 25°C 25°C 37°C 26kDa 25°C 37°C 25°C 37°C 25°C 30 25 20 15 10 S I S I S Non-fused sjIPNS I S I S I S NusA-sjIPNS I S NusA I S I S GST-sjIPNS I S I GST 106 Chapter Results Fig. 4.11 (ctd.) (c) Induction temperature Soluble protein fractions kDa 115 83.0 M C2 37°C 30°C 28°C 25°C 22°C 20°C 18°C 15°C 49.4 37kDa 34.6 29.0 20.4 % of soluble sjIPNS expressed in E. coli BL21(DE3) at different induction temperatures % of soluble sjIPNS expression 35 30 25 20 15 10 37°C 30°C 28°C 25°C 22°C 20°C 18°C 15°C Induction temperature (°C) 107 Chapter 4.2.2 Results Fusion protein Another approach that has achieved success in producing soluble heterologous proteins in E. coli is the use of fusion partners (Section 2.8.2). This has been attributed to the high levels of expression and highly soluble natures of the fusion partner proteins used (Smith et al., 1988; Davis et al., 1999). However, studies have reported that some fusion partners are much better solubilizing agents than others (Kapust and Waugh, 1999; Wang et al., 1999; Tatsuda et al., 2001). In this Section, studies were undertaken to investigate the efficiencies of two fusion partners, GST and NusA, in overcoming the low solubility of sjIPNS. Both GST and NusA have been reported to improve the solubility of some foreign proteins that would otherwise be expressed as insoluble aggregates (Ray et al., 1993; Nygren et al., 1994; Bill et al., 1995; Chang et al., 1997; Davis et al., 1999). To carry out this investigation, sjIPNS was subcloned into fusion vectors, pGK and pET43.1a, that carried genes coding for Schistosoma japonicum GST and E. coli NusA respectively. The detailed plasmid maps of these expression vectors are shown in Appendix III. 4.2.2.1 PCR amplification of sjIPNS to create suitable flanking restriction enzyme sites for subcloning into pGK and pET43.1a fusion vectors The creation of GST-sjIPNS and NusA-sjIPNS fusion constructs requires the cloning of sjIPNS gene downstream to the 3’ ends of the coding genes of the respective fusion partners. Detailed examination of pGK and pET43.1a vector sequences revealed that the ATG start codon of sjIPNS could be precisely inserted in frame with the GST and NusA coding sequences via the use of a BamHI restriction site located near the 3’ends of both fusion protein genes (Fig. 4.12). For the 3’ cloning site, the same XhoI restriction site was chosen for the construction of both GST-sjIPNS and NusA-sjIPNS fusion constructs. pET-SJ plasmid carrying the sjIPNS gene (Section 4.1.2.2) was used for the subcloning experiment. However, Chapter Results the flanking regions of sjIPNS gene in pET-SJ not harbor the selected restriction enzyme sites intended for subcloning. Therefore, PCR amplification was used to create the BamHI and XhoI restriction enzyme sites in the flanking regions of sjIPNS using specially designed primers OL187 and OL188. The sequences of the primers were shown in Appendix II and the optimal reaction conditions used in PCR amplification were the same as that specified in Table 3.5. The ~1 kb amplified BamHI/XhoI flanked sjIPNS product obtained from the PCR reaction was resolved by agarose gel electrophoresis as shown in Fig. 4.13a. The primers designed were very specific since only one distinct amplified product of the correct size was obtained. The amplified product was purified from the agarose gel and sequencing was performed to ensure that the insert corresponds to sjIPNS gene. 4.2.2.2 Subcloning of sjIPNS into pGK and pET43.1a fusion vectors A schematic diagram depicting the cloning strategy of sjIPNS into the two fusion vectors is shown in Fig. 4.12. The sjIPNS PCR product was subsequently subcloned into pGEM-T Easy vector (Promega) (Clark, 1988). BamHI and XhoI double digestion was performed to release the cloned gene insert for ligation to the corresponding sites in pGK and pET43.1a vectors. The resultant recombinant pGK and pET43.1a fusion constructs carrying the sjIPNS gene were named pGST-SJ and pNUSA-SJ respectively. Restriction enzyme digestion was done to affirm that the recombinant constructs contained the cloned gene inserts (Fig. 4.13b). Sequencing of pGST-SJ and pNUSA-SJ using selected sets of primers (Appendix II) was carried out to further confirm the identity of the cloned gene. Electropherograms showing the partial sequenced region (nucleotide 71 to 155) of sjIPNS in both constructs are presented in Fig. 4.14. Repeat sequencing of the forward and reverse strands of the cloned sjIPNS in pGST-SJ and pNUSA-SJ showed no random gene mutations have been incorporated during the amplification and subcloning processes. Chapter Results Fig. 4.12 Construction of recombinant sjIPNS fusion vectors. Schematic diagrams showing the subcloning strategy to construct recombinant fusion vector (a) pGST-SJ and (b) pNUSA-SJ. PCR amplification of sjIPNS sjIPNS pET-SJ 6300bp With primers OL187 and OL188 BamHI Xhol sjIPNS Cloned into pGEM®-T Easy vector BamHI/ Xhol (a) (b) tac GST T7 BamHI BamHI BamH1/ Xhol BamHI/ Xhol Xhol Xhol 7275bp 5189bp BamH1 sjIPNS 6179bp pGST-SJ Xhol BamH1 Xhol sjIPNS 8265bp pNUSA-SJ 110 Chapter Fig. 4.13 Results Gel electrophoresis of PCR amplified sjIPNS containing BamH1/Xho1 flanking ends and restriction enzyme digestion of recombinant fusion vectors. (a) The amplified sjIPNS product (~1kb) with BamH1/Xho1 flanking ends was separated by gel electrophoresis in lane 2. Lane is the PCR control reaction without the addition of DNA template. Lane shows the λHindIII DNA marker. (b) Gel electrophoresis showing the results for the restriction enzyme digestion to confirm that the recombinant pGST-SJ and pNUSA-SJ contain the sjIPNS insert. Lanes and show the λHindIII DNA marker. Lanes and show the products of BamH1 digested pGK and pGST-SJ whereas lanes and show the products of BamH1 digested pET43.1a and pNUSASJ. (a) 23.1 9.4 6.6 4.4 2.2 2.0 ~1kb 0.5 (b) 23.1 9.4 6.6 4.4 2.2 2.0 6.2kb 5.2kb 23.1 9.4 6.6 4.4 2.2 2.0 8.3kb 7.3kb 0.5 0.5 111 Fig. 4.14 Sequence confirmation of cloned sjIPNS in recombinant pGK and pET43.1a fusion vectors. Detailed diagram showing the relative position of IPNS insert with respect to the promoter and ribosome binding site (RBS) in (a) pGK and (c) pET43.1a. (b) The electropherograms showing the partial sequenced regions (nucleotide 71-155) of the cloned sjIPNS gene in recombinant pGST-SJ and pNUSA-SJ fusion vectors are presented in (i) and (ii) respectively. tac promoter lac operator (a) RBS translation start site BamHI ATG GGATCC GST CCTAGG XhoI IPNS insert CTCGAG GAGCTC IPNS insert CTCGAG GAGCTC Nucleotide 71-155 (b) (i) sjIPNS in pGST-SJ (ii) sjIPNS in pNUSA-SJ Nucleotide 71-155 T7 promoter (c) lac operator translation start site BamHI RBS ATG GGATCC NusA CCTAGG XhoI Chapter Results 4.2.2.3 Expression of GST-sjIPNS and NusA-sjIPNS fusion proteins in E. coli BL21(DE3) The recombinant fusion plasmids, pGST-SJ and pNUSA-SJ, were transformed into E. coli BL21(DE3), thereafter the recombinant cultures were induced at 37°C and 25°C to examine the solubilizing effects of both fusion partners on sjIPNS. At the same time, pGK and pET43.1a vector carrying the GST and NusA coding sequences respectively, were also transformed into E. coli BL21(DE3) to be used as controls for the expression studies under the same conditions. The induction and harvesting of recombinant cultures for cell-free extract preparations were performed as described in Section 3.2.9. GST and NusA proteins are 26kDa and 55kDa respectively, hence the expected sizes of GST-sjIPNS and NusA-sjIPNS fusion proteins are ~63kDa and ~92kDa. In recombinant vector pNUSA-SJ, sjIPNS is subcloned downstream of the NusA coding sequencing and the transcription of the NusA-sjIPNS fused gene product is under the control of T7-promoter (Fig. 4.14). In pET-SJ, sjIPNS is similarly cloned downstream of the T7-promoter but is expressed as a non-fused protein (Fig. 4.2). Hence, comparison of the expression of sjIPNS in pNUSA-SJ and pET-SJ can reveal the effect of NusA-fusion on sjIPNS expression in E. coli. The expression results of non-fused sjIPNS under the control of T7-promoter in E. coli BL21(DE3) have already been discussed in Section 4.2.1. Here, the results were again presented alongside with the expression results of fused NusA-sjIPNS protein in the tabulated chart for comparison (Fig. 4.11b). At 37°C and 25°C, majority of the non-fused sjIPNS was expressed in the insoluble fractions. Densitometric scanning results showed that ~29-33% of non-fused sjIPNS was expressed in the insoluble fractions at both temperatures. Interestingly, when sjIPNS was fused to NusA, an estimated to 3-fold reduction in the synthesis of sjIPNS in E. coli was observed. Only about 10-16% of NusA-sjIPNS fusion protein was observed in the insoluble protein fractions (Fig. 4.11b). Nonetheless, observable amounts of soluble NusA-sjIPNS fusion protein up to ~8% of the total soluble proteins was obtained at 25°C. The expression of NusA Chapter Results control protein verified the high solubility nature of NusA protein as the protein was located mainly in the soluble protein fraction at 37°C and 25°C (Fig. 4.11bII). The expression of fused GST-sjIPNS is under the control of tac-promoter instead of the T7-promoter used in the expression of non-fused sjIPNS, hence this precluded the direct comparison of the two sets of results. Clearly the fused GST-sjIPNS was predominantly associated with the insoluble fractions (Fig. 4.11bIII) yielding up to ~28-30% of insoluble protein. Interestingly, GST protein itself was highly soluble (up to ~30% of total soluble proteins) at both 37°C and 25°C (Fig. 4.11bIV). However, despite the high solubility of GST, it did not help to solubilize sjIPNS when sjIPNS was fused to it. 4.2.3 Further lowering of induction temperature Lowering of induction temperature is one of the commonest methods used to improve the expression of soluble proteins (Table 2.6). It remains an attractive method to explore in our efforts to increase the solubility of sjIPNS, although lowering of induction temperature from 37°C to 25°C only resulted in a marginal ~2-3% increase in the amounts of soluble sjIPNS obtained (Section 4.2.1). This is so as the ranges of induction temperatures successfully used to enhance the solubility of expressed proteins were found to vary from 42°C to 15°C as evident from the examples listed in Table 2.6. This suggests that each protein may have a preferred temperature range to enable soluble protein production. A noteworthy point from surveying through the examples in Table 2.6 is that temperatures as low as 15°C was used to improve the soluble expression of three different proteins under the control of T7-promoter. Consequently, 37°C to 25°C may not be an effective temperature range for the expression of sjIPNS in the soluble form. Thus, the expression of sjIPNS under the control of T7-promoter in pET-SJ vector construct was carried out in E. coli BL21(DE3) over a broader spectrum of induction temperatures, ranging from 37°C, 30°C, 28°C, 25°C, 22°C, 20°C, 18°C and 15°C. Chapter Results The soluble fractions obtained at 37°C to 15°C were shown in the SDS-PAGE in Fig. 4.11c. As observed, no overexpressed ~37kDa sjIPNS band was seen in lanes 3-6, representing soluble protein fractions obtained at temperatures from 37°C to 25°C. Unexpectedly, the progressive reduction of temperature from 25°C to 18°C caused a dramatic increase in the amounts of soluble sjIPNS obtained, from ~10% at 22°C to the highest at 18°C which yielded ~30% of soluble sjIPNS. Further reduction to 15°C did not improve sjIPNS soluble expression instead a slight decrease in the amounts of soluble sjIPNS (~16% of the total soluble proteins) obtained was observed. Hence, the experimental results showed that the effective temperature range for high-level production of soluble sjIPNS is between 20°C and 18°C, improving its soluble expression by ~5-6 times compared to the level obtained at 25°C. This is interesting as sjIPNS exhibits a much steeper temperature dependent solubility phenomenon than the rest of the IPNS isozymes studied so far. 4.2.4 Analysis of combining the temperature effect with the study of host strain and sjIPNS fusion proteins The results obtained thus far showed that significantly higher levels of soluble sjIPNS proteins were expressed only when the induction temperature was further reduced to 18°C. However, expression of sjIPNS in E. coli BLStar host and its fusion to GST and NusA respectively only resulted in low levels of soluble sjIPNS being produced when the expression was conducted at 37°C and 25°C. Hence, it will be interesting to compare the effect of reducing the induction temperature on the expression of soluble sjIPNS under the different biophysical conditions. Effect of induction temperature on the expression of soluble sjIPNS in E. coli BLStar host. The expression results of sjIPNS in E. coli BL21 host discussed in Section 4.2.3 were presented here together with the sjIPNS expression results in E. coli BLStar host at 37°C, 30°C, 28°C, 25°C, 22°C, 20°C and 18°C for comparison. The respective soluble protein Chapter Results fractions analyzed by SDS-PAGE are shown in Fig. 4.15a. A similar trend of sjIPNS soluble expression was observed in E. coli BLStar when induction temperature was lowered beyond 25°C, >23% of soluble sjIPNS was expressed between 20°C and 18°C, as evident from the prominent protein bands in lanes and of Fig. 4.15aI. It seems that E. coli BLStar host was also able to express a high level of soluble sjIPNS when the temperature was further reduced. Therefore, the improvement of soluble protein production through lowering temperatures probably occurs via a similar pathway common to both of the E. coli host strains. Effect of induction temperature on the expression of soluble sjIPNS as NusA-sjIPNS and GST-sjIPNS fusion proteins. The lowering of induction temperatures beyond 25°C to 18°C only resulted in marginal improvement in the level of soluble NusA-sjIPNS fusion protein from 8% to 11% (Fig. 4.15bI). This is in contrast to the progressive increment observed for the expression of non-fused sjIPNS at 22°C to 18°C (lanes 7-9 of Fig. 4.15aI). Both the expression of NusA-sjIPNS and non-fused sjIPNS were under the control of T7-promoter, hence it appeared that the fusion of sjIPNS to NusA protein seem to be inhibitory towards the solubilizing effect of lowered induction temperature. Conversely, a marked enhancement in the yield of soluble GST-sjIPNS fusion proteins was observed at 20°C and 18°C. A prominent protein band corresponding to ~18-20% of soluble GST-sjIPNS fusion proteins was detected in the respective soluble protein fractions obtained at these two temperatures. However, the increase in soluble production of GST-sjIPNS at 20°C and 18°C was not as drastic as that observed in the expression of non-fused sjIPNS at the same temperatures. This probably reflects the different strengths of the tac- and T7-promoters used in the expression of the fused GST-sjIPNS and non-fused sjIPNS protein respectively. T7-promoter has been reported to be a stronger promoter then tac-promoter. Summary of the optimization expression studies of soluble sjIPNS. As shown in Table 4.1, at induction temperatures between 37°C and 25°C, a low level of soluble sjIPNS protein was expressed regardless of the E. coli host strains and the fusion expression used. However, at a much reduced induction temperature of 18°C, major improvements in the yield of soluble Chapter Results Fig. 4.15 Expression studies of sjIPNS under the control of various parameters at different induction temperatures. (a) SDS-PAGE analysis of the expression of sjIPNS in E. coli BLStar and E. coli BL21(DE3) using pET-SJ vector construct at 37°C, 30°C, 28°C, 25°C, 22°C, 20°C and 18°C. The soluble protein fractions obtained from sjIPNS expression in E. coli BLStar and E. coli BL21(DE3) are shown in lanes 3-9 of I and II respectively. The protein standards of various molecular sizes (kDa) and the soluble protein fractions of the respective E. coli strains without the pET-SJ vector were loaded into the first and second lane of both gels. The arrows indicate the positions of sjIPNS expressed. The percentages of soluble sjIPNS expressed in the respective fractions were determined using densitometric scanning. II. BL21(DE3) I. BLStar kDa 115 83.0 kDa 115 83.0 49.4 49.4 37 kDa 37 34.6 kDa 34.6 29.0 29.0 20.4 Decreasing temperatures Decreasing temperatures % of soluble sjIPNS expressed in the different E. coli strains at various temperatures % of sjIPNS expression 35 30 E. coli BLStar E. coli BL21(DE3) 25 20 15 10 Induction temperature (°C) 117 Chapter Results Fig. 4.15 (ctd.) (b) SDS-PAGE analysis of the expression of sjIPNS as NusA-fused (I) and GST-fused (II) proteins at various induction temperatures. The respective soluble protein fractions expressed at 37°C, 30°C, 28°C, 25°C, 22°C, 20°C and 18°C were loaded into lanes to of both gels. The protein standards of various molecular sizes (kDa) and the soluble protein fractions derived from the E. coli BL21(DE3) control culture were loaded into lanes and respectively of both gels. The arrows indicate the positions of sjIPNS expressed. The percentages of soluble sjIPNS were determined and plotted in the chart shown below. I. NusA-sjIPNS kDa II. GST-sjIPNS 100 kDa 92kDa 75 50 115 83.0 63kDa 49.4 37 34.6 25 29.0 Decreasing temperatures Decreasing temperatures % of fused sjIPNS expression % of soluble NusA-sjIPNS and GST-sjIPNS expressed at various temperatures 25 20 NusA-sjIPNS GST-sjIPNS 15 10 Induction temperature (°C) 118 Chapter Results sjIPNS were obtained except when sjIPNS was expressed as NusA-sjIPNS fusion protein. In summary, it appears that temperature is the key factor in influencing the levels of soluble sjIPNS production in E. coli. Table 4.1 Summary on the results of sjIPNS soluble expression under different conditions in E. coli. Factors examined Host strain Fusion protein 4.2.5 Soluble sjIPNS expressed as % of total soluble proteins at induction temperatures of 37°C-25°C 18°C BL21(DE3) ≤5% 25-31% BLStar ≤5% 24-28% Non-fused ≤5% 25-31% NusA-sjIPNS ~5-8% 8-11% GST-sjIPNS [...]... expression of NusA-sjIPNS and non-fused sjIPNS were under the control of T7-promoter, hence it appeared that the fusion of sjIPNS to NusA protein seem to be inhibitory towards the solubilizing effect of lowered induction temperature Conversely, a marked enhancement in the yield of soluble GST-sjIPNS fusion proteins was observed at 20 °C and 18°C A prominent protein band corresponding to ~18 -20 % of soluble... recombinant vector pNUSA-SJ, sjIPNS is subcloned downstream of the NusA coding sequencing and the transcription of the NusA-sjIPNS fused gene product is under the control of T7-promoter (Fig 4.14) In pET-SJ, sjIPNS is similarly cloned downstream of the T7-promoter but is expressed as a non-fused protein (Fig 4 .2) Hence, comparison of the expression of sjIPNS in pNUSA-SJ and pET-SJ can reveal the effect of NusA-fusion... Results 4 .2. 2.3 Expression of GST-sjIPNS and NusA-sjIPNS fusion proteins in E coli BL21(DE3) The recombinant fusion plasmids, pGST-SJ and pNUSA-SJ, were transformed into E coli BL21(DE3), thereafter the recombinant cultures were induced at 37°C and 25 °C to examine the solubilizing effects of both fusion partners on sjIPNS At the same time, pGK and pET43.1a vector carrying the GST and NusA coding sequences... induction temperature on the expression of soluble sjIPNS as NusA-sjIPNS and GST-sjIPNS fusion proteins The lowering of induction temperatures beyond 25 °C to 18°C only resulted in marginal improvement in the level of soluble NusA-sjIPNS fusion protein from 8% to 11% (Fig 4.15bI) This is in contrast to the progressive increment observed for the expression of non-fused sjIPNS at 22 °C to 18°C (lanes 7-9 of. .. transformed into E coli BL21(DE3) to be used as controls for the expression studies under the same conditions The induction and harvesting of recombinant cultures for cell-free extract preparations were performed as described in Section 3 .2. 9 GST and NusA proteins are 26 kDa and 55kDa respectively, hence the expected sizes of GST-sjIPNS and NusA-sjIPNS fusion proteins are ~63kDa and ~92kDa In recombinant... the insoluble fractions Densitometric scanning results showed that ~29 -33% of non-fused sjIPNS was expressed in the insoluble fractions at both temperatures Interestingly, when sjIPNS was fused to NusA, an estimated 2 to 3-fold reduction in the synthesis of sjIPNS in E coli was observed Only about 10-16% of NusA-sjIPNS fusion protein was observed in the insoluble protein fractions (Fig 4.11b) Nonetheless,... being produced when the expression was conducted at 37°C and 25 °C Hence, it will be interesting to compare the effect of reducing the induction temperature on the expression of soluble sjIPNS under the different biophysical conditions Effect of induction temperature on the expression of soluble sjIPNS in E coli BLStar host The expression results of sjIPNS in E coli BL21 host discussed in Section 4 .2. 3... activities of non-fused and GST-fused sjIPNS were comparable, 0. 72 and 0.67 units/mg Chapter 4 Results Fig 4.16 Purification of sjIPNS from recombinant E coli pGST-SJ/ BL21(DE3) Purification of sjIPNS through high affinity column chromatography Lane 2 shows the soluble protein fractions of control E coli BL21(DE3) The soluble protein fractions overexpressing GST-sjIPNS is shown in lane 3 Lane 4 and 5 show... NusA-fusion on sjIPNS expression in E coli The expression results of non-fused sjIPNS under the control of T7-promoter in E coli BL21(DE3) have already been discussed in Section 4 .2. 1 Here, the results were again presented alongside with the expression results of fused NusA-sjIPNS protein in the tabulated chart for comparison (Fig 4.11b) At 37°C and 25 °C, majority of the non-fused sjIPNS was expressed in the... levels of soluble sjIPNS production in E coli Table 4.1 Summary on the results of sjIPNS soluble expression under different conditions in E coli Factors examined Soluble sjIPNS expressed as % of total soluble proteins at induction temperatures of 37°C -25 °C 25 -31% ≤5% 24 -28 % Non-fused ≤5% 25 -31% ~5-8% 8-11% GST-sjIPNS 4 .2. 5 ≤5% NusA-sjIPNS Fusion protein BL21(DE3) BLStar Host strain 18°C . hence the expected sizes of GST-sjIPNS and NusA-sjIPNS fusion proteins are ~63kDa and ~92kDa. In recombinant vector pNUSA-SJ, sjIPNS is subcloned downstream of the NusA coding sequencing and. subcloning into pGK and pET43.1a fusion vectors The creation of GST-sjIPNS and NusA-sjIPNS fusion constructs requires the cloning of sjIPNS gene downstream to the 3’ ends of the coding genes. recombinant pGST-SJ and pNUSA-SJ contain the sjIPNS insert. Lanes 1 and 4 show the λHindIII DNA marker. Lanes 2 and 3 show the products of BamH1 digested pGK and pGST-SJ whereas lanes 5 and 6