Figure 1.
Cloning of the coding sequence of ALDH3A1 into the expression vectors.
(A) Construction of the pMAL/hALDH3A1 vector. The HindIII/EcoRI fragment of the PCR product containing the coding region of human ALDH3A1 was inserted into the HindIII and EcoRI sites of the pMAL vector. (B) Construction of the pET-26b (+)/hALDH3A1 vector. The NdeI/XhoI fragment of the PCR product containing the coding region of human ALDH3A1 was inserted into the NdeI and XhoI sites of the pET-26b(+) vector.
Table 1.
Description of the chaperone plasmids used in the study.
Figure 2.
ALDH3A1 heterologous expression through the pMAL-c2X expression system.
SDS-PAGE pattern showing induction of ALDH3A1 expression at (A) 37°C and (B) 25°C. Samples were subjected to SDS-PAGE and stained with Coomassie blue. Lane 1, whole cell lysate prior IPTG induction; Lane 2, whole cell lysate 6 hours after IPTG induction; Lane 3, insoluble fraction; Lane 4: soluble fraction, respectively, 6 hours after IPTG induction. The arrow indicates the position of ALDH3A1/MBP protein.
Table 2.
Comparison of different E.coli strategies for production of soluble recombinant ALDH3A1.
Table 3.
Purification of MBP-tagged recombinant human ALDH3A1 from E. coli.
Figure 3.
ALDH3A1 heterologous expression through the pET-26b(+) expression system.
Induction at (A) 37°C, (B) 25°C and (C) 18°C. Samples were subjected to SDS-PAGE and stained with Coomassie blue. T0: Total cell extract form bacterial culture prior of protein induction, T3: Total cell extract 3 hours after induction, T6: Total cell extract 6 hours after induction, CE: Crude extract of lysed cells 6 hours after induction, IM: Insoluble matter of lysed cells 6 hours after induction. The arrow indicates the position of ALDH3A1/6xHis protein.
Figure 4.
ALDH3A1 heterologous expression through autoinduction with the pET-26b(+) expression system.
Samples were subjected to SDS-PAGE and stained with Coomassie blue. 37°C, 25°C and 18°C: different induction temperatures, CE: Crude extract of lysed cells 9 hours after inoculation, IM: Insoluble matter of lysed cells 9 hours after inoculation. The arrow indicates the position of ALDH3A1/6xHis protein.
Figure 5.
Co-expression of ALDH3A1 with molecular chaperones.
(A) Co-expression of pG-KJE8/pGro7 plasmid’s chaperones, (B) Co-expression of pTf16 plasmid’s chaperones, (C) Co-expression of pKJE7/pG-Tf2 plasmid’s chaperones. Samples were subjected to SDS-PAGE and stained with Coomassie blue. T0: Total cell extract form bacterial culture prior of protein induction, T3: Total cell extract 3 hours after induction, CE: Crude extract of lysed cells 3 hours after induction, IM: Insoluble matter of lysed cells 3 hours after induction. The recombinant proteins expression is presented in the boxes.
Figure 6.
Protein expression and purification of recombinant MBP fused ALDH3A1.
SDS-PAGE analysis at various stages of purification of recombinant MBP-fused ALDH3A1 using amylose resin chromatography (Coomassie blue staining). T0: Total cell extract form bacterial culture prior of protein induction, T6: Total cell extract 6 hours after IPTG induction, IM: Insoluble matter of lysed cells 6 hours after IPTG induction, CE: Crude extract of lysed cells 6 hours after IPTG induction, Elution fractions: purified recombinant ALDH3A1 eluted from amylose resin column. WB: western immunoblotting of purified recombinant ALDH3A1/MBP. The arrow indicates the position of the MBP-fused ALDH3A1 recombinant protein at approximately 92 kDa.
Figure 7.
Protein expression and purification of recombinant his-tagged ALDH3A1.
SDS-PAGE analysis at various stages of purification of recombinant his-fused ALDH3A1 using Ni-affinity chromatography (Coomassie blue staining). T0: Total cell extract form bacterial culture co-expressing pG-KJE8 along with ALDH3A1 prior to IPTG induction, T6: Total cell extract 6 hours after IPTG induction, CE: Crude extract of lysed cells 6 hours after IPTG induction, IM: Insoluble matter of lysed cells 6 hours after IPTG induction, In: Input of the column, Fl: flowthrough part, W: wash part, Elution fractions: purified recombinant protein eluted from Ni-NTA column. WB: western immunoblotting of purified recombinant ALDH3A1/6xHis. The arrow indicates the position of the recombinant his-tagged ALDH3A1 at approximately 51 kDa.
Table 4.
Purification of his-tagged recombinant human ALDH3A1 from E. coli.