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FIGURE 11.2
Microtubule destruction by overexpression of TBCD or TBCE in cultured cells. HeLa cells
were transfected with constructs engineered for the expression of GFP alone (as a control;
A-D), GFP-TBCD (E-H), or GFP-TBCE (I-L). Microtubules are shown in red, detected with
either an anti-
a
-tublin antibody (B, F, and J) or an anti-
b
-tubulin antibody (D, H, and L).
From
Bhamidipati et al. (2000)
depends on the tubulin-specific chaperone: TBCA, TBCB, and TBCC, for example,
can be expressed in
E. coli
in good yield as biologically active proteins. The preferred
expression vector for these cloned cDNAs is one belonging to the pET series (Invi-
trogen Inc.), in which expression is driven from an inducible T7 promoter. Unfortu-
nately, although TBCD and TBCE can also be expressed in
E. coli
, the recombinant
protein is completely insoluble and cannot be recovered as biologically active
material. Resort must therefore be made to eukaryotic host/vector systems. In the
case of TBCE, the biologically active protein can be expressed and purified from
insect sf9 cells via infection with recombinant baculovirus. A number of commer-
cially available vectors and kits are available for this purpose. Although TBCD
can also be expressed as a soluble protein in insect sf9 cells, the yield is relatively
poor and we have not found it possible to purify the protein in active form. The rea-
son for this finding is not clear, but it may reflect the absence of some posttransla-
tional modification that is required for activity. In any event, we have found that
expression of TBCD in cultured human cells via the use of recombinant adenovirus
