T4 DNA LIGASE: DETERMINING THE OPTIMAL SYSTEM FOR INDUSTRIAL EXPRESSION AND PURIFICATION
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Date
2005-09
Department
Hood College Biology
Program
Biological and Environmental Science
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Abstract
Molecular biology enzymes are necessary tools for the
generation of recombinant DNA molecules. Making these
enzymes available at the lowest possible cost will enable
molecular biology laboratories to increase production. In
order to achieve this, molecular biology enzyme supply
companies must determine ways to produce these enzymes at
the lowest possible cost. The production of T4 DNA ligase
was evaluated using E. coli, B. megaterium, and the
baculovirus expression systems. Expression and sonication
studies were performed to optimize enzyme yield. Final
optimal conditions were used to generate 1L cell pellets
for purification. Protein assays confirm a higher yield of
T4 DNA ligase from the baculovirus system than from E. coli
or B. megaterium. It was hypothesized that the specific
activity of purified T4 DNA ligase from each system would
be similar. A novel technique using the Agilent 2100
Bioanalyzer was evaluated to determine ligase activity.
The assay results indicate all three expression systems
produced ligase of similar activity. The cost analysis, in
conjunction with the yield indicates E. coli is the best
system for recombinant T4 DNA ligase production at the 1L
scale.