Mark Gerard Obukowicz

Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of pseudomonads using Tn5.

The delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 was cloned into Tn5 and the resulting Tn5-tox element transposed from a vector plasmid into the chromosome of six corn-root-colonizing strains of Pseudomonas fluorescens and Agrobacterium radiobacter. Chromosomal integration of the tox gene maximized stability and minimized the potential for horizontal transfer of the tox gene to other bacterial species. Expression of the tox gene was demonstrated by Western blot analysis and by toxicity against larvae of the tobacco hornworm (Manduca sexta). The method described illustrates how a given gene can be stably integrated into the chromosome of diverse bacterial species.

Secretion and export of IGF-1 in Escherichia coli strain JM101

SummaryThe processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (Processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.

IS50L as a non-self transposable vector used to integrate the Bacillus thuringiensis delta-endotoxin gene into the chromosome of root-colonizing pseudomonads

Insertion sequence IS50L of transposon Tn5 was used as a non-self transposable vector to integrate the delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 into the chromosome of two corn-root colonizing strains of Pseudomonas fluorescens (112-12 and Ps3732-3-7). A DNA fragment containing the KmR gene from Tn5 and tox was inserted into an IS50L element (IS50L-tox) contained on a suicide plasmid. Transposition of IS50L-tox into the chromosome of P. fluorescens 112-12 and Ps3732-3-7 occurred by selecting for KmR transconjugants and supplying transposase in cis from a linked IS50R element. A frameshift mutation in the transposase gene of the IS50L-tox element was also constructed to decrease the likelihood that suppression or a spontaneous reversion at the UAA (ochre) termination codon of IS50L would create an active transposase. The inability of IS50L-tox to transpose further minimizes the potential for horizontal gene transfer of the tox gene to other bacterial species. Expression of the Tox protein in strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).

A chromosomal expression vector for Escherichia coli based on the bacteriophage Mu

A new Escherichia coli expression vector with increased stability was developed based on bacteriophage Mu. Unlike traditional expression vectors, the vector described herein is chromosome based rather than existing as an autonomously replicating plasmid. The chromosomal location resulted in extreme stability of the vector even in the absence of selective pressure. Both replication and heterologous protein synthesis could be induced by temperature shift. Expression of the heterologous gene was controlled by the Mu middle promoter and was dependent on the presence of the transactivator, Mor, of the Mu middle promoter. Four proteins, beta-galactosidase, chloramphenicol acetyltransferase, porcine somatotropin and human growth hormone, were made from this vector at levels ranging from 5 to 20% of total cell protein. Expression from the middle promoter was highest when inductions were done in rich media. The expression of some genes varied in different strains.

Expression, purification, and in vivo activity of atrial natriuretic factor prohormone produced in Escherichia coli.

Atrial muscles of the heart are known to produce polypeptide hormones called atrial natriuretic factors (ANF) which have potent diuretic and hypotensive action. These hormones are synthesized as a larger protein precursor called pro atrial natriuretic factor or proANF which contains the biologically active ANF sequences at its C-terminus. Rat proANF (representing amino acids -1 to 128 of the coding sequence) was expressed in a soluble form in Escherichia coli. A simple purification procedure was developed which consists of boiling E. coli cell extracts in 1 M acetic acid and subjecting the supernatant to reversed-phase HPLC. The effect of intravenous administration of the purified recombinant proANF on mean arterial blood pressure was examined. The displacement dose-response curves obtained demonstrated that proANF exhibits similar, albeit less potent, physiological activity than ANF.