Engineering Variant Forms of Alpha-1-antitrypsin

Abstract

Alpha-1-antitrypsin deficiency (A1AD) is a genetic condition that can lead to early onset emphysema around 30 to 40 years of age, and with some versions of the condition, it can also lead to other complications such as liver cirrhosis much earlier in life. A1AD affects around 100,000 people in the U.S., with less than 10 percent of those affected being properly diagnosed. Diagnosis is complicated and time-consuming, and the symptoms of A1AD mimic those of asthma which may delay diagnosis and treatment. This suggests a need for new diagnostic tests.
The “normal” form of the alpha-1-antitrypsin protein (i.e., the M form) is expensive but otherwise readily available commercially for purchase and study. However, the mutated versions (i.e., variants of the protein associated with A1AD or total absence of the protein) are not available, which leads to the need to produce these mutated forms. To do so, we acquired plasmid DNA containing genes for both the M form and the Z form which is the variant most often associated with A1AD. These plasmids will allow expression of the M and Z forms in mammalian cells, but this is expensive. Thus, to enable the variants to be produced in non-pathogenic bacteria (which represents a much cheaper alternative), these plasmids were then cut using appropriate restriction enzymes such that the genes for the M and Z forms could be isolated and then inserted into a plasmid to allow for propagation in chemically competent E. coli cells. Herein, we report on the progress to date in the production of normal and variant forms of A1AT.
We then verified that the cells had the plasmid in them and ran an arabinose study to determine the optimal concentration to produce the most amount of A1AT protein. Extraction of the protein was done by affinity chromatography. Then, testing was done by a Bradford assay to determine the concentration.