Effects of the PT region of EngD and HLD of CbpA on solubility, catalytic activity and purification characteristics of EngD-CBD(CbpA) fusions from Clostridium cellulovorans.
Chimeric proteins combining the catalytic N-terminal region of native EngD with its proline-threonine-threonine (PT) linker region, hydrophilic domain (HLD) and cellulose binding domain (CBD) of cellulose binding protein A (CbpA) from Clostridium cellulovorans were constructed, expressed, and analyzed. The chimeric proteins with CBD(CbpA) all demonstrated strong affinity to Avicel. The chimeric protein with the PT region of EngD and the HLD had the best catalytic activity and the highest estimated percentage of soluble protein amongst the chimeric proteins. Native EngD and two of the chimeric proteins (EngD-PT-HLD-CBD and EngD-CBD) were purified and their characteristics analyzed. Their binding affinities to Avicel as well as their enzymatic activities against various substrates were found to be consistent with the results we saw from protein lysate samples, which was good binding to Avicel but a decrease in solubility and catalytic activities in chimeric proteins without PT and/or HLD. The reasons for these are discussed. These fusion proteins may be important in applications, such as immobilization to solid cellulose substrate for purification of proteins and enrichment/aggregation of protein complexes.
Yeh
Michael
M
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Craig
Scott
S
Lum
May-Ghee
MG
Foong
Frances C
FC
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046974:Clostridium cellulovorans / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D002480:Cellulase