Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.

Simultaneous regulation of plasmid replication and heterologous gene expression in Escherichia coli.

An expression plasmid in which plasmid DNA replication and heterologous gene expression can be simultaneously regulated was constructed to avoid derepression prior to induction. This was achieved by placing a pBR322 origin of replication immediately downstream of an anthranilate synthase-human epidermal growth factor fusion gene (trpE-hEGF), both under the control of the promoter from the tryptophan biosynthetic operon. Regulation of plasmid copy number ensured tight repression of the trp promoter prior to induction. Upon induction, plasmid copy number increased up to six-fold and the fusion protein accumulated to approximately 12% of total cell protein. Induction experiments with a series of plasmid derivatives with sequentially lower copy numbers revealed that accumulation levels of the TrpE-hEGF fusion protein post-induction correlated well with plasmid copy number. Plasmid constructs where the native trp promoter had been replaced by derivatives deleted of the attenuator resulted in high levels of hEGF accumulation in the tryptophan-free medium prior to induction. Nevertheless, up to two-fold increase in TrpE-hEGF accumulation levels were obtained using the constructs lacking the attenuator compared to those bearing the native trp promoter.

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

The 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.

Analysis of transcription from the trfA promoter of broad host range plasmid RK2 in Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa.

Reverse transcriptase mapping has been used to analyze transcription from the trfA promoter of broad host range plasmid RK2. The results show that trfA operon mRNA has the same 5' end in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli. The strengths of wild-type and mutant trfA promoters, which differ by defined base substitutions, have been compared and the positions of their transcriptional start sites determined. While these base substitutions do not alter the transcriptional start site, they do have marked effects on promoter strength which are broadly similar in each of the host species. A single base pair substitution, which lies in the region corresponding to the E. coli promoter consensus, brings about a large reduction in gene expression while the introduction of a second mutation, at a locus outside this region, has no further effect on promoter strength. The results indicate that these Pseudomonas species possess an RNA polymerase which recognizes the same region of the trfA promoter as that utilized by E. coli RNA polymerase. Within the limits of these observations it is clear that the trfA operon is transcribed from a single promoter which can function efficiently in diverse species, a property which may be important for its broad host range.

Characterization and incompatibility properties of ROM- derivatives of pBR322-based plasmids.

We have characterized a copy number mutant of the pBR322-based plasmid pWT111. A single nucleotide transversion in loop II' of RNAI results in an eightfold increase in plasmid copy number. Removal of the rom coding region from pWT111 cop results in a further sixfold increase in copy number. We present evidence that ROM is involved in the strong incompatibility effect seen between pMB1 and ColE1 type plasmids.

Use of a modified Escherichia coli trpR gene to obtain tight regulation of high-copy-number expression vectors.

It has been found that with high-copy-number vectors utilising the Escherichia coli trp promoter the amount of repressor protein produced from the single chromosomally located trpR gene is inadequate for tight repression to be obtained. An attempt has been made to overcome this problem by inserting the trpR gene in cis into the expression vector. This proved unsuccessful because transcription from the trp promoter of such a plasmid could not be induced with 3,beta-indole acrylic acid, probably because the trpR gene is autogenously regulated. However, it was found that when the natural trpR promoter was replaced with a relatively weak constitutive promoter a useful self-repressible vector could be formed. A modified trpR gene of this type has been used to obtain tightly controlled expression of human interferon-beta (IFN-beta) from a vector having a copy number of 400. Tight regulation is particularly important in this case as IFN-beta is highly toxic to the E. coli cell.

Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator.

Two DNA fragments which contain the Escherichia coli tryptophan promoter operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the "attenuator peptide" coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the "attenuator peptide" SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.

Influenza antigenic determinants are expressed from haemagglutinin genes cloned in Escherichia coli.

A gene sequence for the fowl plague virus (FPV) haemagglutinin molecule has been inserted into a bacterial plasmid such that its transcription is under the control of a promoter derived from the tryptophan operon. Such plasmids direct the synthesis of a protein that reacts specifically with antisera to FPV haemagglutinin. Evidence is also presented that in some cases DNA inserted at the HindIII site of pBR322 is expressed.