Isopropyl-β-d-thiogalactosidase (IPTG)-Inducible Tyrosine Phosphorylation of Proteins in E. coli

The post-translational modification of proteins by tyrosine phosphorylation plays a critical role in signal transduction. As a consequence, this modification can dramatically alter the protein’s activity or stability. To study the biological and biochemical properties of these proteins in vitro, a source for obtaining an adequate amount of phosphorylated protein is essential. In many instances, the phosphoprotein is expressed in cell systems such as baculovirus/insect cells or eucaryotic cell lines expressing an appropriate kinase. However, the use of these methods is relatively time consuming. Alternatively, the recombinant proteins can be phosphorylated in vitro with purified kinase, but the cost associated with these procedures is often prohibitive. Phosphorylation of mammalian proteins expressed in bacteria has been described (2,3,7). In a commercially available system, Epicurian Coli TKX1 Competent cells (Stratagene, La Jolla, CA, USA), a mammalian tyrosine kinase encoding plasmid under the control of the procaryotic trp promoter is used to phosphorylate target proteins expressed in E. coli. If the target protein has an affinity tag or is fused to glutathione-S-transferase (GST) or maltose binding protein (MBP), then the production and purification of large amount of this protein is relatively rapid and easy. However, the use of the trp promoter is rather cumbersome. To induce the expression of the kinase, the cells must be transferred to M9 minimal medium and treated with indole acrylic acid. To make this method of generating phosphoproteins more “user friendly”, we generated a plasmid that controls the expression of the tyrosine kinase with the hybrid tac promoter (4). This plasmid, pTac-elk, encodes a rat tyrosine kinase (elk) (5) whose expression is induced by treatment with isopropyl-β-D-thiogalactosidase (IPTG). The plasmid also encodes tetracycline resistance, making it easy to co-select with many other conventional ampicillin-resistant vectors. The plasmid harbored in the E. coli strain TKX1 (Stratagene) was isolated. The trp promoter was replaced by tac promoter from pGEX-2T (Amersham Pharmacia Biotech, Piscatatway, NJ, USA) by a two-step PCR method (shown schematically in Figure 1). In brief, a pair of primers was designed containing an initiation ATG in the middle and homology to the tac promoter sequence of pGEX2T upstream and homology to elk coding region downstream (plus-tac: 5′-ttcacacaggaaacagtattcATGGAAGCTGTCCGGGAGTTTGCC-3′; minus-tac: 5′-GGCAAACTCCCGGACAGCTTCCATgaatactgtttcctgtgtgaa-3 ′; the lowercase letters indicate the sequence from pGEX2T, and the uppercase letters indicate the sequence from elk). The PCR product containing lac operator and tac promoter from pGEX2T were generated by primer up-tac (5 ′-gcgcccatggcatcataacggttctggc-3 ′) and minus-tac, and a NcoI site (underlined) was added to its 5 ′ end. The second PCR product containing the elk segment was amplified with primer down-elk (5′-ctttgcacaccaggttgc-3 ′) and plus-tac. The resulting two PCR products were purified, mixed together along with the flanking primers up-tac and down-elk, and reamplified. This PCR product was restricted with NcoI and ApaLI and gel purified. This fragment was used to replace the NcoI and ApaLI fragment of the progenitor plas mid (containing the trp promoter). The integrity of the resulting plasmid was confirmed by DNA sequencing. To test the induction of the pTac-elk plasmid, a fragment of Stat3α (amino acids 570–770), containing tyrosine 705, was amplified by PCR and cloned into pGEX2T. JM 109 (recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi∆(lacproAB) F′[traD36 proAB+ lacI q