Production of an Antibody for the Lwf Protein in Drosophila melanogaster

Laura Korthauer
Marquette University
Mentor: Dr. Edward Blumenthal

Lot’s wife (lwf1) is a mutation in the CG33968 gene in Drosophila melanogaster.  Flies with lwf1 die a few days after eclosing as adults.  Mutated flies seem to have difficulty moving food from their crops to the midgut.  In order to understand the nature of the Lwf protein missing in mutated flies, immunostaining will be performed.  Before doing this, an antibody must be raised against the Lwf protein.  The goal of this study was to express and purify a piece of the Lwf protein so that an antibody can be created for use in further experimentation.

Amino acids 21-115 of the lwf gene were found to be the best match for raising an antibody against the Lwf protein.  After using PCR to amplify the chosen region of the lwf gene, the insert was subcloned into a plasmid vector.  The ligation of insert and vector was confirmed by various restriction digests.  The plasmid was then transfected into BL21 cells to express the protein.  The protein was expressed in detectable levels, and it was found in the insoluble fraction.  Attempts were made to make the protein soluble by varying growth conditions, but it is presumed to be in inclusion bodies and is unable to be made soluble.  The protein goes into solution in urea and will be purified under denaturing conditions on a Ni/NTA column.  It will then be produced in larger quantities so that an antibody can be raised against it. 
 
 

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