In vitro mutagenesis and overexpression of the @entity526 trpA gene and the partial characterization of the resultant XXXX synthase mutant alpha-subunits.

@entity19592 @entity535 @entity1132 @entity2340 @entity17241 @entity7783 @entity5189 @entity526
A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the @entity526 @entity2340 synthase. A random single base pair saturation mutagenesis procedure (Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 229, 242-247) was applied in vitro to subcloned fragments of the trpA gene, which codes for this polypeptide. Mutagenesis plasmid vectors were constructed containing three fragments of the trpA gene which together code for about half of the total @entity535 residues of the alpha-subunit. The vectors were constructed such that each strand of each trpA fragment could be altered. These trpA fragments were mutagenized in vitro (using @entity17241 , @entity5189 , @entity7783 , and @entity19592 ), and several thousands of mutants have been isolated. Thirty-two mutants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the alpha-subunit) have been sequenced. Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alterations resulted in single @entity535 substitutions. Selected mutant trpA fragments were subcloned into an overexpression vector in which the trpA gene is controlled by the tac promoter and is inducible by @entity1132 . The kinetics and extent of induction show that after 22 h of induction, the wild-type alpha-subunit constituted about 30% of the total protein. A simple one-step purification procedure for the alpha-subunit is described in which 15 mg of alpha-subunit can be obtained from 200 ml of fully induced cultures. The mutant trpA genes were induced for mutant alpha-subunit expression, and an initial examination of their properties in crude extracts was performed. Of the 17 mutant proteins examined, most were overproduced to levels comparable to that for the wild-type alpha-subunit. An analysis of the apparent stability, beta 2-subunit-activating activity, and intrinsic activity of this group of mutant alpha-subunits suggests that many @entity535 alterations have no apparent effect; there is a variety of novel functional defects; and a sequence located near residues 28 through 54 may be particularly critical for the normal folding of the polypeptide.
