We next test the expression conditions of PV in pET22b and pRSETb vectors. PV in two different vectors (pRETb-PV and pET22b-PV) were expressed in LB medium (Fig. 3-5). When the OD500 reached to 0.8, the bacteria were induced with 0.1 mM, 0.5 mM or 1 mM IPTG. As shown in Fig. 3-5, the protein greatly expressed after 3 hours induction and same level of expression were observed after IPTG induction overnight. For pRSETb-PV vector, the best IPTG concentra- tion for PV expresion is 0.5 mM and the protein expression level decreased when IPTG concen- tration increase to 1 mM. However, the best concentration of IPTG for the expression PV in pET22b vector is different from that in pRSETb vector. As shown in Fig. 3-5, 1 mM IPTG can in- duce the highest level of PV expression in pET22b vector. Compared with the expression levels of PV with different IPTG concentration and in two vectors, we found that the best PV expres- sion condition is pET22b vector and induced at 1 mM IPTG for 3 hr or overnight.
Figure 3-5. The expression of PV by E. coli.
pRESTb-PV (left top) and pET22b-PV (left bottom) plasmids were transferred into bacteria BL21- DC3-plysS strain and were further expressed in LB-medium with IPTG induction.
3.2.3.2 Early procedure for PV purification (Protocol 1)
Fig. 3.6 shows the early procedure for PV purification. BL21 plysS bacteria with pET22b- PV transformation were expressed in 2 liter LB medium. When the optical density (OD) reached to 0.8, the bacteria were induced with either 1 mM IPTG. The protein greatly expressed after 3 hours induction. High amount of proteins were expressed in pET22b vectors after cultured at 37 ˚C overnight. After that, the proteins were purified followed by Henzl’s 172 protocol with modifi- cations. SDS-PAGE and DNA agarose gel were used to monitor the PV purification. To purify this protein, the bacteria with PV expresion were lysated by French press or sonication. As shown from the SDS-PAGE in Fig. 3-7, PV mainly exists in the supernatant after spinning down at 13000 rpm for 10 min. The purification of PV took the advantage of high thermal stabilities of this pro- tein. The supernatant of bacterial lysate were incubated at 85 ˚C for 5-20 min. After heating and centrifugation, most of the PV was in the supernatant while other proteins were denatured and precipitated by high temperature (Fig. 3-7 A). We then ran two set of FPLC equipped with Hi- Trap Q column to further purify PV. The PV in the first round of FPLC were eluted by 10 mM Tris/HCl, pH7.6, 3 mM EGTA and 1 M NaCl. As shown in the SDS-PAGE in Fig. 3-7 B, the fractions 12 and 13 contains PV with 95% purity. However, DNA agarose gel indicates that fractions 12 and 13 contains DNA and RNA (Fig. 3-7 B), although major DNA peak is shown in fraction 15 and 16. To further purify PV, we collected fraction 12 and 13, added 6 mM CaCl2 and then injected into FPLC. As shown from Fig. 3-7, fraction 4-8 contains PV with high purity. After purified by second round of FPLC, PV with more than 99 % purities were obtained (Fig. 3-7).
Figure 3-6. Schematic representation of initial strategies for PV purification (Protocol 1). PV is expressed in E. coli after IPTG induction. Then, the bacteria were breaked by sonication and/or French press. The supernatant of bacteria lysate were incubated in a water bath at 85 ˚C for 5-20 min. The bacteria were further purified by two round of FPLC with different elution buffer. For the first round of FPLC, buffer A contains 3 mM EGTA in HEPES buffer at pH 7.2, buffer B contains 3 mM EGTA, 1 M NaCl in HEPES buffer at pH7.2. All the fractions of PV were further purified with second round of FPLC with 3 mM CaCl2 instead of EGTA in both buffer A and buffer B.
Figure 3-7. Purification of PV with two round of FPLC.
The purification of PV using stretegies shown in Figure 3-6 monitored by SDS-PAGE (A, C and B (top)) and agarose gel (B (bottem)). A. Expresion of PV induced by IPTG and purification of PV
by frech press and heat incubation. B. Different fractions from first round FPLC purification with HP Q colume, monitored by SDS-PAGE (top) and DNA agarose gel (bottem); C. Different frac- tions from second run FPLC purification equated with Q HP column.
3.2.3.3 Structural characterization of purified PV variants
We next scan the UV absorbance spectra of PV and it mutants. Since pavalbumin wild type doesn’t have trptophan and tyrosine, the UV absorbance peak is mainly from Phe at 259 nm. As shown from Fig. 3-8 A, PV wild type has the maximum peak at 259 nm, which is con- sistent with literature reported carp PV absorbance spectrum.183 PV variants with tryptophan showed the UV absorbance peak at 280 nm and a sharp peak at 285-290 nm (Fig. 3-8B, C, D), which is also consistent with literature reported absorbance spectrum of PV with F103W muta- tion184. UV absorbance spectrum also indicates that purified PV variants do not have DNA or RNA contamination.
We further perform MALDI-mass analysis to verify our purified PV (Fig. 3-9). The mo- lecular weight of the purified protein is 11776.8 kDa, which is consistent with the calculated value of PV (11776 kDa).
Figure 3-8. UV spectra of PV.
UV spectra of PV wild type (A) and its variants: PVF103W (ProCA31) (B), PVS56DF103W (ProCA32) (C), and PVS60DF103W (ProCA33) (D).
Figure 3-9. Mass spectra of PV.
The expected MW of PV is 11776 kDa. The MALDI measured molecular weighte of PV is 11776.8 kDa.
3.2.3.4 Further optimization and simplification of for PV purification (Protocol 2)
Previously, we are able to obtain the recombinant PV and its variants using heating combined with two round of FPLC in the present or absent of Ca2+ (Fig. 3-6). Using this method, we are able to obtain PV with 99% purity. However, lots of proteins were lost during these two sets of FPLC. In order to improve the yield of PV, remove DNA contamination more efficiently and simplify the purification procedure, six major modifications were made based on previous purification protocol (Fig. 3-6). 1). We added additional step before FPLC for DNA precipitation. We added 3 % streptomycin sulfate and incubated at 4 C overnight. After spin down at 13000 rpm for 10 min the supernatant were incubated at 85 C for 5 min. After spin down, the super- natant was dialysis against 10 mM Tris/HCl at pH 8.0 before injecting into FPLC column. 2). We increased the buffer pH 7.5 to 8.0. Since the Q column is anion exchange column, increasing the pH of buffer improves the binding of PV to the column. 3). We found that our protein can be eluted out from Q column at 200 mM NaCl, therefore, we decrease the percentage of buffer B (containing 1 mM NaCl) during elution from 70% to 25% of buffer B. 4). The flow rate of FPLC was decreased from 5 ml/min to 2 ml/min to improve the binding of the pavalbumin to Q col- umn. 6). We use 9 column volumes to let buffer B slowly increase from 0% to 25% (Fig. 3-10). The very slow increase of salt gradient further facilitates the separation between PV and DNA. After FPLC remaining small fragment of DNA can be further removed by concentrating protein using through 3 kDa membrane.
The FPLC prufication curve is shown in Fig. 3-11A. The UV absorbance at 280 nm from FPLC detector shows four peaks during buffer B elution. Each peak were collected and then tested by UV spectroscopy, SDS PAGE and agarose gel. As show from Fig. 3-11A, the third frac-
tion (28-30) has a single band at 11 kDa, which is consistent with the molecular weight of PV. UV spectrum of this frection is the same as the PV F103W mutants in the old methods in 3.2.3.2. The first and second peaks do not have any protein and the last peak contains protein with a molecular of 30 kDa. DNA and UV absorbance data show that the peak 1 and peak 4 has a lot of DNA/RNA because these frections have high obsorbance at 260 nm. The final yield of this protein is about 50-70 mg per liter bacteria expression in LB medium. Taken together, this new method simplified PV purification procedure with much higher yield compared with the early purification methods described in section 3.2.3.2.
Figure 3-10. The FPLC program for ProCA3 variants purification.
After binding to the column, the unbounded component were first washed with 7 column vol- ume (CV) of buffer A (10 mM HEPES buffer at pH 8.0). Then, buffer B (10 mM HEPES buffer, 1 M NaCl at pH 8.0) concentration was increase to 25 % within 7 CV. We further wash the column with 25% of buffer B for 2 CV and then increase to 100 % of buffer B of 1 CV to wash out every component bounded to the column. At last, the column was requilibriated with 100% buffer A for 5 CV.
Figure 3-11. FPLC fractions, SDS-PAGE and UV absorbance spectrum analysis of ProCA3 purifi- cation with improved protocol.
Only one round of FPLC are required in this new purification method. There are four major peaks during FPLC purification (A). SDS-PAGE results (B) show that the fractions 28-32 contain ProCA3 with more than 99% purity. UV spectrum of fraction 28-32 (C) shows the typical spec- trum of ProCA3, which has the maximum absorbance at 280 nm and a shoulder peak at 285 nm. The UV absorbance at 260 nm is lower than that at 280, indicating the DNA concentration in these fractions are very low.
Figure 3-12. Conformational analysis of ProCA3 variants in the presence Ca2+ or EGTA by CD. The ProCA3 variants shows typical helix structure in CD and the secondary structures of proCA3 variants do not change in the presence Ca2+ or EGTA at 10 mM Tris buffer at pH 7.2.