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1.
The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered. 相似文献
2.
Biotination of proteins is a post-translational modification that requires a folded acceptor domain. We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo. We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli. Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product. This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein. The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease. Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes. These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo. 相似文献
3.
The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane. 相似文献
4.
The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli. 相似文献
5.
Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase. 总被引:14,自引:0,他引:14
The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed. 相似文献
6.
Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter
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Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme beta-lactamase, leading to efficient expression of the fusion protein in E. coli. The beta-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located beta-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of beta-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E. coli cells expressing the beta-lactamase AIDA-I fusion protein remained physiologically intact. 相似文献
7.
H Kolmar E Ferrando F Hennecke J Wippler H J Fritz 《Journal of molecular biology》1992,228(2):359-365
A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide. 相似文献
8.
9.
The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose. 相似文献
10.
11.
A protein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of beta-lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat-modifiable phenotype of gonococcal P.II. 相似文献
12.
K Fujimori S Fukuzono N Kotomura N Kuno N Shimizu 《Bioscience, biotechnology, and biochemistry》1992,56(12):1985-1990
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. 相似文献
13.
We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla). In the first fusion gene blahi, TEM1 beta-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer. The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds. 相似文献
14.
15.
An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening. 相似文献
16.
Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272. 相似文献
17.
Random oligonucleotide fragments were designed and amplified by PCR and fused with the activating domain of pGAD424 to construct a random peptide library. The DNA fragment encoding beta-lactamase was fused with the binding domain of pGBT9(+2). Subsequently, using yeast two-hybrid system we found two positive clones encoding peptides P1 and P2 that have the ability to bind beta-lactamase in vivo. The genes encoding P1 and P2 were cloned into pGEX-4T-1. GST-peptide fusion proteins were expressed in Escherichia coli and isolated by glutathione-Sepharose 4B affinity chromatography. Finally, P1 and P2 were cleaved from the fusion protein with thrombin and purified by ultrafiltration. Inhibition assay of peptides with beta-lactamase in vitro indicated that only P1 has the ability to inhibit beta-lactamase. 相似文献
18.
The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli. 相似文献
19.
Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase. 总被引:1,自引:0,他引:1
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The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli. 相似文献
20.
Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase. 总被引:5,自引:5,他引:0
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J Luirink T Watanabe H C Wu F Stegehuis F K de Graaf B Oudega 《Journal of bacteriology》1987,169(5):2245-2250
A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells. 相似文献