In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha-subunits.

D L Milton,M L Napier,R M Myers,J K Hardman

doi:10.1016/s0021-9258(18)66610-4

Abstract

A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the Escherichia coli tryptophan 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 amino acid 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 nitrous acid, formic acid, hydrazine, and potassium permanganate), 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 amino acid 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 lactose. 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 amino acid 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.

Highlights

In Vitro Mutagenesis and Overexpressionof the Escherichia coli trpA Gene andthe Partial Characterization ofthe Resultant Tryptophan Synthase Mutant a-Subunits”
During the construction of the mutagenesis vectors, emphasis was placed on maintainingorcreating unique restriction sites for the mobilization of each trpA fragment into and out of the mutagenesis vectors during the mutagenesis protocol and for the replacement of the wild-type trpA fragment with the mutant trpA fragment in the overexpression vector, ptactrpA, while stillmaintaininga proper reading frame, Details of these constructions are given in the Miniprint section
Asystematic treatment of theentire gene is planned,our initial efforts are directed toward the examination of this region of the polypeptide chain because of its importance in the initial step(s) of the proposed folding pathway

Summary

Present address

Since only 10% of the transformants obtained contains mutant trpA fragments, it was necessary to apply a selection step This selection utilizes a urea-formamide gradient (DNA-melting) polyacrylamide gel system and recovers trpA fragmentscontainingbp differences from the wild-type fragments. This procedure requires the presence of the GC clamp (a 300-bp GC-rich sequence),which remains helical over the entire range of formamide denaturant used in the gradient, and permits the recovery of all sequence-altered trpA fragments of variable melting properties [29]. During the construction of the mutagenesis vectors, emphasis was placed on maintainingorcreating unique restriction sites for the mobilization of each trpA fragment into and out of the mutagenesis vectors during the mutagenesis protocol and for the replacement of the wild-type trpA fragment with the mutant trpA fragment in the overexpression vector, ptactrpA, while stillmaintaininga proper reading frame, Details of these constructions are given in the Miniprint section

RESULTS AND DISCUSSION

LQ sites-altered

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