Figure 1.
Inclusion bodies formation of XynA expressed from plasmids pHsh-xynA2 and pET-xynA2.
A–B. Electronic microscopy observation of inclusion bodies in the pHsh-xynA2 transformed E. coli cells (A) and pET-xynA2 transformed E. coli cells (B), ultrathin section. C. SDS-PAGE analysis of XynA expressed from pET-xynA2. Lanes: M, protein marker; 1, total protein of E. coli containing pET-20b(+) without target gene; 2, total protein and 3, soluble protein of E. coli containing pET-xynA2 grown at 37°C; 4, total protein and 5, soluble protein of E. coli containing pET-xynA2 grown at 20°C.
Table 1.
Plasmids.
Figure 2.
SDS-PAGE analysis of inclusion bodies produced from the fused genes.
Lanes: M, protein markers; 1, whole cell protein of pHsh transformed E. coli; 2–3, expression of XarB from pHsh-xarB transformed E. coli, showing soluble protein (lane 2) and insoluble protein (lane 3) fractions; 4–5, expression of fused gene of xarB and xynA2 in plasmid pHsh-xar-xyn, showing soluble protein (lane 4) and insoluble protein (lane 5) of E. coli; 6, inclusion bodies of fused XarB and the C-terminus of XynA N-terminus of XynA; 7, inclusion bodies of fused XarB and the C-terminus of XynA. Arrows indicate the bands o f recombinant proteins.
Figure 3.
SDS-PAGE analysis for the expression of recombinant proteins in pHsh-ex.
A. Soluble expression of XynA from pHsh-ex-xynA2; lanes: M, protein markers; 1, total protein of E. coli containing pHsh-ex; 2, total protein and 3, soluble protein of E. coli containing pHsh-ex-xynA2; 4, periplasmic protein obtained by cold osmotic shock; 5, XynA purified by ion exchange chromatography. B. Comparison of fused protein expressed from plasmids pHsh-xar-xyn and pHsh-ex-xar-xyn; lanes: M, protein markers; 1, total protein of E. coli containing pHsh; 2–4, total protein, soluble protein and insoluble protein of E. coli containing pHsh-xar-xyn, respectively; 5, total protein of E. coli containing pHsh-ex; 6–8, total protein, soluble protein and insoluble protein of E. coli containing pHsh-ex-xar-xyn, respectively. Arrows indicate the bands of recombinant proteins.
Table 2.
Purification of recombinant xylanase from E. coli harboring pHsh-ex-xynA2.
Table 3.
Conditions to produce soluble protein from xynA2.
Figure 4.
The expression of soluble xylanase and cultivation profile of E. coli harboring pHsh-xynA2, pET-xynA2, pHsh-ex-xynA2 and pHsh-ex-xynA–m1.
A. SDS-PAGE analysis of the soluble xylanase; Lanes: M, protein marker; 1–4, soluble protein of E. coli containing pHsh-xynA2, pET-xynA2, pHsh-ex-xynA2, and pHsh-ex-xynA-m1, respectively. B. Cultivation profiles of recombinant E. coli cells after induction. Symbols: cells harbored plasmid pHsh-xynA2 (-▪-), pET-xynA2 (-▴-), pHsh-ex-xynA2 (-•-) or pHsh-ex-xynA-m1 (-♦-).
Figure 5.
The effect of initial cell density for induction on the production of recombinant periplasmic protein in the E. coli cells harboring pHsh-ex-xynA2.
Symbols: -□- cell density; -▪- xylanase activity.
Figure 6.
Immuno-detection of σ32 in E. coli K-12 cells carrying Hsh vector, and over-expression of XynB in pHsh.
A. The variation of σ32 concentrations in cells subjected to heat-shock induction. Recombinant cells carrying pLac-xynB or pHsh-xynB were grown to OD600 = 0.7 at 30°C then transferred into a 42°C water-bath incubator; samples were withdrawn at various timepoints and cell densities were determined; cells harvested from samples were re-suspended in the volume of 1.5×SDS sample buffer adjusted to a equivalent cell density of OD600 = 30, and 10 µl of lysates were loaded for immuno-blotting. B. Detection of σ32 in 6 transformants grown at 30°C; E. coli cells were transformed by pLac-xynB or pHsh-xynB, single colonies were grown to OD600 = 3 (about 6 h), and cells were harvested from 1 ml culture and re-suspended in 0.1 ml of lysis buffer (1.5×SDS), and 15 µl samples were loaded on SDS-gel for immuno-blotting after incubated in boiling water bath for 10 min. C. Expression of XynB from pLac-xynB and pHsh-xynB, Lanes: M, protein markers; 1–3, gene expression tests for pLac-xynB, showing intracellular protein of recombinant cells without induction, and with IPTG induction and heat-shock induction, respectively; 4 and 5, intracellular protein of pHsh and pHsh-xynB transformed cells induced by heat-shock, respectively. Arrows indicate the bands of recombinant proteins.
Table 4.
Primers.