+ Site Statistics
References:
54,258,434
Abstracts:
29,560,870
PMIDs:
28,072,757
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

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



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



Journal of Biological Chemistry 261(35): 16604-16615



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.

(PDF emailed within 1 workday: $29.90)

Accession: 040392175

Download citation: RISBibTeXText

PMID: 3023357


Related references

In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant a-subunits. The Journal of Biological Chemistry 261: 604-15, 1986

In vitro mutagenesis of the escherichia coli trpa gene and the expression and properties of corresponding mutant trpa alpha subunits. Federation Proceedings 45(6): 1929, 1986

Enzymatic properties of mutant escherichia coli tryptophan synthase alpha subunits obtained from in vitro mutagenesis of the trp a gene. FASEB Journal 4(7): A1760, 1990

Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli. Biochemical and Biophysical Research Communications 289(2): 568-572, 2001

Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits. Journal of Biological Chemistry 266(30): 20205-20212, 1991

Structure and folding of mutant escherichia coli tryptophan synthase alpha subunits. Journal of Cellular Biochemistry Supplement (9 PART B): 95, 1985

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits. Archives of Biochemistry and Biophysics 292(1): 34-41, 1992

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli. Biochemical and Biophysical Research Communications 300(1): 29-35, 2002

Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits. Journal of Bacteriology 173(6): 1886-1893, 1991

Substrate interactions with the alpha-subunit of the Escherichia coli tryptophan synthase. A study of the activity of mutant alpha-subunits. Archives of Biochemistry and Biophysics 181(2): 428-437, 1977

Crystallization and X-ray crystallographic studies of wild-type and mutant tryptophan synthase alpha-subunits from Escherichia coli. Molecules and Cells 19(2): 219-222, 2005

A molecular characterization of spontaneous frameshift mutagenesis within the trpA gene of Escherichia coli. Dna Repair 6(2): 177-189, 2006

Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase. Journal of Biological Chemistry 270(47): 28177-28182, 1995

Tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase. Enzymatic and urea stability properties. Journal of Biological Chemistry 270(30): 17712-5, 1995

Enzymatic properties of mutant Escherichia coli tryptophan synthase a-subunits. The Journal of Biological Chemistry 266: 205-12, 1991