High-level expression of human insulin-like growth factor II in Escherichia coli.

A gene encoding mature human insulin-like growth factor II (IGF-II) was constructed from the modified IGF-II cDNA sequence and two double-stranded synthetic oligodeoxynucleotide linkers. It was fused to a truncated lacZ gene such that IGF-II was expressed as part of C-terminus of beta-galactosidase. This fused lacZ'-IGF-II gene was under the control of tac promoter and we overproduced the beta-galactosidase-IGF-II fusion protein in the Escherichia coli. The fusion protein formed inclusion bodies inside the cells. The fusion protein was purified from the isolated inclusion bodies and IGF-II protein was obtained from their fusion protein by CNBr cleavage. The released IGF-II was confirmed by its molecular weight as determined by SDS-PAGE and by its ability to bind anti-IGF antibody.     

Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli

To study the role played by acetate metabolism during high-cell-density growth of Escherichia coli cells, we constructed isogenic null mutants of strain W3100 deficient for several genes involved either in acetate metabolism or the transition to stationary phase. We grew these strains under identical fed-batch conditions to the highest cell densities achievable in 8 h using a predictive-plus-feedback-controlled computer algorithm that maintained glucose at a set-point of 0.5 g/l, as previously described. Wild-type strains, as well as mutants lacking the s^s subunit of RNA polymerase (rpoS), grew reproducibly to high cell densities (44–50 g/l dry cell weights, DCWs). In contrast, a strain lacking acetate kinase (ackA) failed to reach densities greater than 8 g/l. Strains lacking other acetate metabolism genes (pta, acs, poxB, iclR, and fadR) achieved only medium cell densities (15–21 g/l DCWs). Complementation of either the acs or the ackA mutant restored wild-type high-cell-density growth. On a dry weight basis, poxB and fadR strains produced approximately threefold more acetate than did the wild-type strain. In contrast, the pta, acs, or rpoS strains produced significantly less acetate per cell dry weight than did the wild-type strain. Our results show that acetate metabolism plays a critical role during growth of E. coli cultures to high cell densities. They also demonstrate that cells do not require the s^s regulon to grow to high cell densities, at least not under the conditions tested. Journal of Industrial Microbiology & Biotechnology (2000) 24, 421–430. Received 12 November 1999/ Accepted in revised form 24 February 2000     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli.

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.     

Effects of La3+ on growth,transformation, and gene expression of Escherichia coli

Rare earth elements have been emitted into the environment largely as fertilizer components. This has caused much fear about whether they would influence our environment, especially on the metabolism and genetics of microorganisms. In this article, the trivalent ion of a rare earth element, lanthanum, was studied for the effects on growth, transformation, and gene expression of Escherichia coli. The results showed that La³⁺ at concentrations from 50 to 150 μg/mL stimulated the endogenic metabolism and ectogenic metabolism, but had few effects on gene expression. La³⁺ at lower concentrations from 0.5 to 30 μg/mL inhibit intensively E. coli-absorbing external DNA, decreasing the transformation efficiency. It is also supported by observations using transmission electron microscopy. Our results are significant in understanding the function of rare earth elements to microorganisms and assessing the risk of application of rare earth compounds.     

Effects of chaperones on mRNA stability and gene expression in Escherichia coli

Effects of chaperones on mRNA stability and gene expression were studied in order to develop an efficient Escherichia coli expression system that can maximize gene expression. The stability of mRNA was modulated by introducing various secondary structures at the 5'-end of mRNA. Four vector systems providing different 5'-end structures were constructed, and genes encoding GFPuv and endoxylanase were cloned into the four vector systems. Primer extension assay revealed different mRNA half-lives depending on the 5'-end secondary structures of mRNA. In addition to the stem-loop structure at the 5'-end of mRNA, coexpression of dnaK-dnaJ-grpE or groEL-groES, representative heat-shock genes in E. coli, increased the mRNA stability and the level of gene expression further, even though the degree of stabilization was varied. Our work suggests that some of the heat-shock proteins can function as mRNA stabilizers as well as protein chaperones.     

Effects of deletion and insertion mutations in the ilvM gene of Escherichia coli.

A plasmid was constructed that carried the ilvG and ilvM genes and the associated promoter and leader regions derived from the K-12 strain of Escherichia coli. The ilvG gene contained a + 1 frameshift mutation that enabled the plasmid to specify acetohydroxyacid synthase II. The plasmid was modified by deletions in the terminus of and within the ilvM gene and by insertions into the ilvM gene. The effects of these modifications on the phenotypes of the plasmids were examined in a host strain that lacked all three isozymes of acetohydroxyacid synthase. Most of the ilvM mutant plasmids so obtained permitted growth of the host strain in the absence of isoleucine but not in the absence of valine. Growth in the presence of valine, however, was very slow. No significant acetohydroxyacid synthase activity could be detected even when the cells were grown in a valine-supplemented minimal medium. It thus appears that, at most, only a very low level of acetohydroxyacid synthase activity occurred with ilvG in the absence of ilvM and that low activity was more effective for acetohydroxy butyrate formation than for acetolactate formation. The ilvM gene product could be formed under the control of the lac promoter in the presence of a plasmid that carried an in-frame gene fusion between lacZ and the downstream portion of ilvG. Extracts from the host strain that contained such an IlvG(-)-IlvM+ plasmid could be combined with extracts from cells that contained one of the IlvG+-IlvM- plasmids to yield acetohydroxyacid synthase activity. Thus, the ilvM and ilvG genes could be expressed independently of each other.     

Effects of l-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGF1). Fed-batch fermentation of E. coli TG1/pAPT-K6Ub-IGF1 resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM L-arginine, and 4 °C was chosen to optimize the refolding of K6Ub-IGF1, and 240 mg/L of denatured K6Ub-IGF1 was refolded with a 32% yield. The positive effect of L-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGF1 from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGF1, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that L-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.     

Expression of secreted insulin-like growth factor-1 in Escherichia coli

The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1 or somatomedin-C) fused to LamB and OmpF secretion leader sequences in Escherichia coli have been investigated. Expression and secretion of IGF-1 was achieved. The major portion of this secreted IGF-1 accumulated in the periplasmic space as insoluble aggregates. A small amount of IGF-1 was found folded in its native conformation in the medium. The lamB and ompF signal sequences were fused to the 5' coding sequence of IGF-1. Fusion of the lamB signal sequence directly to IGF-1 (lamB-IGF-1) resulted in accumulation of 16-20 micrograms/A550/ml of correctly processed IGF-1 in the periplasmic space. The processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in an E. coli strain bearing a prlA4 mutation. Amino acid sequence analysis of IGF-1 secreted into the periplasm and exported into the medium confirmed the precise removal of the LamB or OmpF signal sequence. IGF-1 synthesized in E. coli was demonstrated to be active in a cell proliferation bioassay.     

Quinolone-resistant mutations of the gyrA gene of Escherichia coli

Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.     

Up-promoter mutations in the lpp gene of Escherichia coli.

The promoter of the gene for the major outer membrane lipoprotein, the most abundant protein in Escherichia coli, is considered to be one of the strongest promoters in E. coli. The nucleotide sequences of the -10 and the -35 regions of the lpp promoter were altered in a step-wise manner to conform to their respective consensus sequences by synthetic oligonucleotide-directed site-specific mutagenesis. The mutated promoters were then fused to the lacZ gene to measure promoter activity. The beta-galactosidase activity increased approximately 1.9 and 2.4 fold when the -10 region (AATACT) was altered to TATACT(P1) and TATAAT (consensus sequence; P2), respectively. Similarly, it increased approximately 1.2 and 4.2 fold, when the -35 region (TTCTCA) was altered to TTCACA(R1) and TTGACA (consensus sequence; R2), respectively. When the mutations at the -10 and -35 regions were combined, the overall improvement of the promoter activity for R2-P1 was 4.0 fold over that of the wild-type promoter, while it was only 2.5 fold for R2-P2. These results indicate that substantial improvement of the promoter activity can be achieved by changing either of the two key regions to their respective consensus sequences. However, the complete conformity to consensus sequences at both regions does not necessarily result in the highest activity. With use of the improved lpp promoter in an expression cloning vehicle pIN-III-ompA, staphylococcal nuclease A was produced at a level of approximately 47% of the total cellular protein.     

RifR mutations in the beginning of the Escherichia coli rpoB gene

In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid β subunit of RNA polymerase. Almost all sequenced RifR mutations occur within the Rif region, encompassing rpoB codons 500–575. A strong Rif^R mutation lying outside the Rif region, which changed Val¹⁴⁶ to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for Rif^R mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong Rif^R mutations were obtained when Val¹⁴⁶ was mutated, and several weak Rif^R mutations were also isolated near position 146. The results define a new, N-terminal cluster of Rif^R mutations, in addition to the classical central Rif region.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.