Part:BBa_K1418012
Cellulase (cenA) generator with 10x His-tag
Protein generator part for chlorophyllase. Part BBa_K118023 was obtained from the parts registry and is the cenA coding sequence encoding Cellulomonas fimi endoglucanase A. In order to be compatible for fusion protein applications, primers were designed to make BBa_K118023 RCF 23 compatible. The product of this PCR reaction was cloned into pSB1C3 and is designated as BBa_K1418010. The double stop codons were then removed via PCR, cloned into pSB1C3 and this construct is designated as BBa_K1418011. Part BBa_K1418011 was then cloned behind a lac inducible promoter and a ribosome binding site (K208010 contains both R0010 and B0034). To aid in protein purification, a 10x histidine tag with two transcriptional terminators (K844000) was cloned in frame on the 3' end to generate the final composite construct, BBa_K1418012.
To test for protein production from the new BBa_K1418012 construct, protein purification using a nickel column was performed. Using methods provided in our "protocols" section, cells containing BBa_K1418012 were grown overnight, pelleted and lysed. After centrifugation, the supernatant was applied to the nickel column. Various samples throughout the purification process were analyzed using SDS-PAGE. The SDS-PAGE below shows results from analysis of cells containing BBa_K1418012.
It can be seen from the SDS-PAGE that a protein product around 45kDa has been purified been purified using the nickel column. Since the expected size of our cellulase construct is 46 kDa, we are confident that we have purified the cellulase enzyme.
To test for enzymatic activity, extracts used in lanes 2-8 of the SDS-PAGE shown above were applied to a carboxymethyl cellulose plate and then stained with Congo Red according to methods detailed in our "protocols" page.
From this result, it can be observed that extracts from the elution fractions (6-8) had more cellulase activity than fractions 2-5 (flow through and washes). The purified cellulase fractions had similar activity as the positive control, which contained 1mg/ml of commercial cellulase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 260
Illegal NotI site found at 1404 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1317
Illegal BamHI site found at 453
Illegal XhoI site found at 815
Illegal XhoI site found at 1064 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 595
Illegal NgoMIV site found at 1520 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 499
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