Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter

To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300 000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lacUV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.     

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.     

Overproduction of biologically-active human nerve growth factor in Escherichia coli.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

Modulation of expression of the human gamma interferon gene in E. coli by site-directed mutagenesis

Plasmids expressing 2 forms of human immune interferon (IFN-gamma) in E. coli have been constructed: 1) pIFNTacI which expresses IFN-gamma with an N-terminal amino acid sequence of met-cys-tyr-cys-gln-, and 2) pIFNTacII which is a derivative of pIFNTacI from which the 9 base pairs (bp) coding for the cys-tyr-cys have been deleted. Quantitation of Western blots showed that approximately 10-fold more IFN-gamma was produced in cells harboring pIFNTacII (7.5% of total cellular protein) as compared to pIFNTacI. The IFN-gamma expressed in E. coli pIFNTacII is biologically active and routinely recoverable at 10(9) units per liter. When examined microscopically, IPTG induced E. coli harboring either plasmid construction contains prominent cytoplasmic inclusion bodies.     

Expression in Escherichia coli of chemically synthesized gene for a novel opiate peptide alpha-neo-endorphin

Chemically synthesized alpha-neo-endorphin gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pKO13. The resulting recombinant DNA was used to transform E. coli cells. Radioimmunoassay for alpha-neo-endorphin in CNBr-treated bacterial cells showed that alpha-neo-endorphin was synthesized at approximately 5 x 10(5) molecules per single E. coli cell. One of the transformants, WA802/p alpha NE2, was used for alpha-neo-endorphin purification. From 10.9 g of wet cells, we isolated 4 mg of chemically pure and biologically active alpha-neo-endorphin.     

Cloning and expression of the cloacin DF13/aerobactin receptor of Escherichia coli (ColV-K30)

A DNA fragment derived from the ColV-K30 plasmid and coding for both sensitivity to cloacin DF13 and Fe3+-aerobactin uptake was cloned into pBR322. The cloned fragment coded for two polypeptides with molecular masses of 74,000 (the cloacin DF13/aerobactin receptor protein) and 50,000 daltons, respectively. When grown with sufficient iron, cells harboring pFS8 (with this fragment) possessed about 10 times as many receptor protein molecules as compared with cells of Escherichia coli (ColV-K30). The synthesis of the receptor protein specified by pFS8, however, was independent of the availability of iron, in contrast to strains harboring the intact ColV-K30 plasmid. Aerobactin was taken up but not synthesized by cells harboring pFS8. No growth occurred when iron-starved cultures of these cells were incubated with Fe3+-aerobactin, suggesting that expression of other ColV-K30-encoded genes is necessary to remove the iron from the Fe3+-aerobactin complex.     

Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.     

Structural and functional stability of IncP plasmids during stepwise transmission by trans-kingdom mating: promiscuous conjugation of Escherichia coli and Saccharomyces cerevisiae

In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes. A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E. coli harboring pAY101, and E. coli carrying a helper plasmid with mob and tra. In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10(-5) to 10(-3) per recipient cell. This E. coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes. The mob and tra genes were trans-acting elements as in an E. coli conjugation system. The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation. DNA analysis indicated that the plasmid pAY101 was transferred from E. coli to S. cerevisiae, and retained its original structure and function in yeast host cells.     

Nucleotide sequence of the lspA gene,the structural gene for lipoprotein signal peptidase of Escherichia coli

The nucleotide sequence of the lspA gene coding for lipoprotein signal peptidase of Escherichia coli was determined and the amino acid sequence of the peptidase was deduced from it. The molecular mass and amino acid composition of the predicted lipoprotein signal peptidase were consistent with those of the signal peptidase purified from cells harboring the lspA gene-carrying plasmid. The peptidase most probably has no cleavable signal peptide. The lspA gene was preceded by the ileS gene coding for isoleucyl-tRNA synthetase and the tandem termination codons of the ileS gene overlapped with the initiation codon of the lspA gene.     

Expression of human salivary alpha-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence

A cDNA fragment coding for human salivary alpha-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 X 10(5) molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as alpha-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.     

Synthesis of fusion and mature murine alpha interferons in Escherichia coli

Four murine interferon-alpha (MuIFN-alpha) genes (alpha 1, alpha 4, alpha 5, alpha 6) were previously identified and characterized. The coding regions of these IFN-alpha genes were inserted into bacterial expression vectors behind the lpp promoter under the control of the lac promoter-operator region, resulting in fusion peptides containing additional N-terminal amino acids (aa). Plasmids coding for the expression of mature IFN-alpha 1 and alpha 5 were also constructed using the same vector system, by inserting a 30-bp synthetic oligodeoxynucleotide, which contains a stop codon for the lpp gene, a ribosome-binding sequence and an ATG start codon for the IFN peptides. The amounts of IFN polypeptides synthesized in Escherichia coli were estimated in the maxi-cell system and their biological activities were measured on mouse and other mammalian cells. The yields of mature IFN produced in this vector were 2 to 4 X 10(6) units/liter; the antiviral activity of the majority of the MuIFNs on human and bovine cells was 100- to 1000-fold lower than on mouse cells. IFN-alpha 4, which contains an internal deletion of 5 aa, showed a lower antiviral activity than other MuIFNs on mouse cells.     

Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses

The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as alpha-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 10(4)-10(5) molecules of alpha-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.     

人α-型肿瘤坏死因子cDNA在大肠杆菌中的高效表达

人α-型肿瘤坏死因子(Tumor Necrosis Factor,TNF-α)可以在体内或体外特异地杀伤多种肿瘤细胞,并同时具有抗病毒感染及免疫调节活性,是一种非常有前途的抗肿瘤及抗病毒生物制剂。最近,国外用基因工程技术将TNF-α基因克隆并在大肠杆菌及酵母细胞中表达成功。目前,人们正致力于提高其在大肠杆菌中表达产量的研究。据此,我们构建了P_L启动子控制的TNF-α cDNA表达载体,使其在大肠杆菌中获得了高水平表达。     

Increased production of aspartase in Escherichia coli K-12 by use of stabilized aspA recombinant plasmid

Recombinant plasmid pYT471, which consists of the aspartase gene (aspA) and the multicopy vector pBR322, was lost from cells of Escherichia coli K-12 at high frequencies in medium in which aspartase was abundantly formed due to release from catabolite repression. This plasmid loss was not completely prevented by the selective pressure of antibiotic addition. To increase the stability of the aspA plasmid, pNK101 (pBR322::aspA-par) was constructed by using the partition locus (par) derived from the low-copy vector pSC101. In E. coli K-12 cells, pNK101 was lost at a frequency as low as 0.4% per cell generation in nonselective medium, whereas pYT471 was lost at a frequency as high as 8.5%. Cells harboring this stable plasmid produced ca. 30-fold more aspartase than did cells harboring the unstable plasmid after 30 cell generations. Thus, we could increase aspartase production by stabilizing the aspA recombinant plasmid.     

Increased production of aspartase in Escherichia coli K-12 by use of stabilized aspA recombinant plasmid.

Recombinant plasmid pYT471, which consists of the aspartase gene (aspA) and the multicopy vector pBR322, was lost from cells of Escherichia coli K-12 at high frequencies in medium in which aspartase was abundantly formed due to release from catabolite repression. This plasmid loss was not completely prevented by the selective pressure of antibiotic addition. To increase the stability of the aspA plasmid, pNK101 (pBR322::aspA-par) was constructed by using the partition locus (par) derived from the low-copy vector pSC101. In E. coli K-12 cells, pNK101 was lost at a frequency as low as 0.4% per cell generation in nonselective medium, whereas pYT471 was lost at a frequency as high as 8.5%. Cells harboring this stable plasmid produced ca. 30-fold more aspartase than did cells harboring the unstable plasmid after 30 cell generations. Thus, we could increase aspartase production by stabilizing the aspA recombinant plasmid.     

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain.

The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy. Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E. coli lpp host. Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe. The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min. Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases. The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface. It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E. coli, while binding to peptidoglycan also occurs over the entire cell surface. The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction. Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell. Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.