Abstract
Well-established genetic manipulation procedures along with a fast doubling time, the ability to grow in inexpensive media, and easy scaleup make Escherichia coli (E. coli) a preferred recombinant protein expression platform. Human alpha-1 antitrypsin (AAT) and other serpins are easily expressed in E. coli despite their metastability and complicated topology. Serpins can be produced as soluble proteins or aggregates in inclusion bodies, and both forms can be purified to homogeneity. In this chapter, we describe an ion-exchange chromatography-based protocol that we have developed involving the use of two anion-exchange columns to purify untagged human AAT from E. coli. We also outline methods that can be used to determine the inhibitory activity of both AAT in cell lysates and purified AAT. Our protocol for the purification of bacterially expressed AAT yields pure and active protein at 6-7 mg/l culture.