Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli.

Abstract

The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the A phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed. The cloned T4 pin gene does not seem to affect the turnover of normal E. coli proteins.The introduction of cloned genes into Escherichia coli bacteria for the purpose of producing large amounts of specific proteins not normally present in E. coli cells has led to an examination of various problems associated with the expression of such cloned genes. Difficulties in obtaining expected levels of expression from genes cloned in bacterial cells may be encountered at the levels of stability of the gene and its vector within the host cell (1, 2), transcription of the cloned gene (3-7), and translation of its mRNA into protein (5,6,8 Difco agar, maltose/L bottom agar is identical to maltose/L top agar. Bacteria that were to be assayed for interferon activity were grown in the presence of the appropriate antibiotic(s) in either M9, which contains, per liter: NH4Cl, 1.0 g; KH2PO4, 3.0 g; Na2HPO4, 6.0 g; NaCl, 5.0 g; MgSO4 7H2O, 0.25 g; CaCl2, 0.015 g; glucose, 2.0 g; supplemented with vitamin BI and Casamino acids to final concentrations of 0.001% and 0.5%, respectively, 2059The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease

“…The effects of pin on various proteins that are usually unstable in E. coli were examined. A marked decrease in the degradation of puromycyl polypeptides was observed in cells in which T4 pin was expressed (38); these results were similar to earlier results with the complete phage (39). In addition, pin increased the stability of cloned mature, human fibroblast interferon in E. coli (38).…”

“…A marked decrease in the degradation of puromycyl polypeptides was observed in cells in which T4 pin was expressed (38); these results were similar to earlier results with the complete phage (39). In addition, pin increased the stability of cloned mature, human fibroblast interferon in E. coli (38). The 6.0 x 104 to 6.0 x 106 ml-' and incubated for 2 h at 30°C with aeration to permit phage multiplication.…”

“…Lambda phage containing the Ots mutation form plaques efficiently at 40°C on bacteria wit'h reduced Lon protease activity; such bacteria include lon [L,utants (14,37) and cells expressing the T4 pin function (36,38 6.0 x 10-. After a lon mutation was transduced into NL2810, the strain (NL2812) was able to support plaque formation by lambda cI857 Ots at 40°C.…”

Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa

“…The two sets of plasmids constructed were transformed into E. coli S17-1(pir) (Simon et al 1983) by electroporation and transformants selected on MHA supplemented with Ap (100 µg mL) and Tc (20 µg/mL). Single colonies were purified and screened by PCR.…”

Isolation and Characterization of the Phage T4 PinA Protein, an Inhibitor of the ATP-dependent Lon Protease ofEscherichia coli

“…Inhibition of protein degradation requires synthesis of T4 proteins made during the first 10 min after infection at 37°C (18 -20). Clones of T4 genes that lead to inhibition of proteolysis in E. coli cells were obtained by Simon and co-workers (19,20), and one specific gene, pinA (proteolysis inhibition A), which resulted in inhibition of abnormal protein degradation, was cloned by Skorupski et al (21). The target of PinA in vivo appears to be the ATP-dependent Lon protease, because E. coli lon ϩ cells expressing a single copy of the T4 pinA gene are phenotypically Lon Ϫ (21).…”