Constructs Used
Cel9B of T. fusca , similarly to cellulases from other
microorganisms, is comprised of a series of recognizable domains,
including a catalytic domain and the carbohydrate binding domains CBM2
and CBM4, as well as the smaller domains of unknown function Eset and
Fn3. A depiction of the structure is presented in Figure 1.
The accession number for Cel9B is Q08166. Cel9B-1.pET26b-1, containing
wild type Cel9B, was previously cloned into plasmid pET26b-1 using
restriction sites Xho I and Nde I (Collmer and Wilson,
1983). A number of truncation mutant forms of Cel9B were generated with
various functional domains removed or retained (Figure 1).For each
truncation mutant, the catalytic domain was retained but each
carbohydrate binding motif was removed to create the mutants M1 (missing
CBM2) and M2 (missing CBM4). Truncation mutants E9B and Fn3 were created
by removing all functional domains except for the catalytic domain and
E9B or Fn3, respectively. Truncation mutants ΔE9B and ΔFn3 lack the
functional domains E9B and Fn3 located between the CBM and the catalytic
domain, respectively. Truncation mutant cd9B contained only the
catalytic domain. M1, M2, cd9B, Fn3, and E9B were PCR amplified using
the primers listed in Table 1. Primers were designed according to
homology to structural domains identified with other cellulases. Cel9B
deletion mutants ΔFn3 and ΔE9B were constructed by PCR amplification of
deletion constructs M2 and M1, respectively. During primer design, a
6His tag was included on the C-terminal portion of the protein. PCR
products were digested with Xho I and Nde I, then religated
to linearized vector pET26 and transformed into DH5α cells. Subclones
containing each truncation mutant were identified by nucleotide sequence
analysis and retransformed into E. coli BL-21 cells for protein
amplification.