EXPRESSION OF MICROTUBULE MOTOR PROTEINS IN BACTERIA FOR CHARACTERIZATION IN IN-VITRO MOTILITY ASSAYS

This chapter describes some of the bacterial expression systems in use, with emphasis on those that have been used to express microtubule motor proteins. It also examines protocols used to obtain active motor proteins from bacteria, assays that can be used for biochemical characterization of motor proteins, and in vitro motility assays. Expression of foreign proteins in bacteria has become a very powerful technique for obtaining large quantities of protein. The DNA sequence, encoding a protein (or a segment of it), is ligated into a unique restriction site in the expression vector with the initiating methionine in the correct reading frame. Standard cloning techniques are used to transform the ligated DNA into bacterial cells, select transformants carrying the DNA insert in the correct orientation, and transfer constructs into appropriate host cells for protein induction. The pET and pGEX bacterial expression systems have been used successfully for the expression of microtubule motor proteins. The pET system is useful for expressing either a nonfusion protein or a fusion protein with only 10 to 13 additional amino acids at the N terminus of the expressed protein. Expression of proteins cloned in PET vectors is under the control of a T7 promoter. © 1993, Elsevier Science Publishers, B.V.