Protein Engineering vol.5 no.3 pp. 279-283. 1992

Overproduction, preparation of monoclonal antibodies and purification of E.coli asparagine synthetase A

Susan K.Hinchman and Sheldon M.Schuster The Department of Biochemistry and Molecular Biology, University of Florida College of Medicine. Box J-245, JHMHC. Gainesville, FL 32610, USA

Introduction Asparagine synthetase (AS) catalyzes the formation of asparagine from L-aspartate, ATP, and a nitrogen source by the following reactions: L-Asp + L-GIn + ATP - L-Asn + L-Glu + AMP + PP: L-Asp + NH4+ + ATP - L-Asn + AMP + PP;

(1) (2)

Asparagine synthetase has been isolated from a variety of prokaryotic and eukaryotic organisms. Ammonia dependent asparagine synthetases, which catalyze only reaction (2), have been identified in prokaryotes such as Lactobacillus arabanosis (Ravel, 1970), Streptococcus bovis (Burchall etal., 1964), Klebsiella aerogenes (Reitzer and Magasanik, 1982) and Escherichia coli (Cedar and Schwartz, 1969a,b). Escherichia coli has two distinct genes for AS, asnA and asnB, which are coded for separately. Asparagine synthetase A catalyzes only reaction (2), whereas AS B performs similarly to the enzyme in eukaryotic organisms, catalyzing both reactions. The nucleotide sequences for both E.coli genes have been determined (Nakamura et al., 1981; Scofield etal., 1990). Asparagine synthetase from a variety of mammalian sources has been purified and shown to catalyze both the glutamine and ammonia dependent reactions (Hongo and Saito, 1981, 1983; Luehr and Schuster, 1985). The ability to use ammonia in addition to glutamine has been shown for a variety of glutamine admidotransferases. As a result of the sequence comparison with © Oxford University Press

Materials and methods Bacterial strains and plasmids All strains were derivatives of E.coli K12: BL21DE3pLys S (F~ ompT, rb", mb") from Studier (Studier and Moffatt, 1986) TG-1 [K12, (lac-pro), supE, thi, hsd5l¥' traD36, proA+B+, /ac/qM15] from Amersham, NM522 [SupE, thi, (lac-proAB) hsd5,{rm~)l¥' proAB. /ac/qZM15] from Stratagene. The plasmid pTZ18R was purchased from Pharmacia LKB Technologies, and pBluescript was purchased from Stratagene. The plasmids pET31FlmlHCA-2 and pET31FlBl were a gift from P.Laipis. pLAsnA was a gift from H.Zalkin. Materials Restriction and modifying enzymes were purchased from Promega, BRL, or New England Biolabs. Immunoblotting reagents were purchased from Sigma. CNBr activated Sepharose 4B was purchased from Pharmacia LKB Technologies. CM279

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In order to explore the structure-function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E.coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E.coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E.coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E.coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step. Key words: antibody production/asparagine synthetase/ Escherichia coli

glutamine amidotransferases, it was proposed that human AS and the E.coli AS B belong to the purF subfamily (Buchanan, 1973; Pruisner and Stadtman, 1973; Hongo and Saito, 1983; Meister, 1985). A common feature of this subfamily is an N-terminal cysteine which is required for activity (Walker et al., 1984; Surin and Downie, 1988). Replacement of the codon for the N-terminal cysteine of human AS with the codon for alanine, by site directed mutagenesis, resulted in a mutant AS protein which lost glutamine dependent activity, but did not affect ammonia dependent activity (Van Heeke and Schuster, 1989). The amino acid sequence of human AS has been aligned with E.coli AS A and E.coli AS B. The resulting alignment showed clear evidence for homology between E.coli AS B and human AS, with 37% of the amino acids identical (Scofield etal., 1990). These results provided evidence that E. coli AS B and human AS may be evolutionarily related. No amino acid sequence similarities were found between E.coli AS A and human AS or between E.coli AS A and E.coli AS B. Asparagine synthetase A was not found to be similar to any proteins currently listed in the various computer databases. This leaves the question of the evolutionary relationship of E. coli AS A open. Since the gene for AS A has been sequenced, and the amino acid sequence deduced, it is possible to look for similar structural motifs among the enzymes available in the protein databases. Such a search has revealed very little useful information. The best approach to understanding the relationship of structure to function is via crystallography or other physical techniques. While a sufficient amount of enzyme has been purified by Cedar and Schwartz to do detailed kinetic analysis (Cedar and Schwartz, 1969a,b), the next stage of investigation will require large amounts of very pure enzyme. Obtaining this goal must be accomplished if we are to be able to assign structural features of E.coli AS A to the various chemical reactions and partial reactions which have been characterized (Cedar and Schwartz, 1969,a,b). Here we report a method for overexpressing E.coli asnA and purifying large amounts of E.coli AS A.

S.K.Hinchman and S.M.Schuster

Construction of pET-A: pET31flBl contains two unique cloning sites, Ndel and BamHl. Cloning of a fragment into the Ndel site allows for proper spacing between the start codon of the target gene and

Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A.

In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overe...
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