Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants.
Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.
Zhang
Dang-Quan
DQ
The Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
Liu
Bing
B
Feng
Dong-Ru
DR
He
Yan-Ming
YM
Wang
Jin-Fa
JF
D021301:Antifreeze Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D018552:Daucus carota / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D021301:Antifreeze Proteins; D017931:DNA Primers; D011994:Recombinant Proteins