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1.
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
2.
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
3.
Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli 总被引:4,自引:0,他引:4
A genetic system for directly synthesizing eukaryotic membrane proteins in Escherichia coli and assessing their ability to insert into the bacterial cytoplasmic membrane is described. The components of this system are the direct expression vector, pYZ4, and the mature beta-lactamase (BlaM) cassette plasmid, pYZ5, that can be used to generate translational fusions of BlaM to any synthesized membrane protein. The beta-subunit of sheep-kidney Na,K-ATPase (beta NKA), a class-II plasma membrane protein, was synthesized in E. coli using pYZ4, and BlaM was fused to a normally extracellular portion of it. The fusion protein conferred ampicillin resistance on individual host cells, indicating that the BlaM portion had been translocated to the bacterial periplasm, and that, by inference, the eukaryotic plasma-membrane protein can insert into the bacterial cytoplasmic membrane. A series of 31 beta NKA::BlaM fusion proteins was isolated and characterised to map the topology of the eukaryotic plasma membrane protein with respect to the bacterial cytoplasmic membrane. This analysis revealed that the organisation of the beta NKA in the E. coli cytoplasmic membrane was indistinguishable from that in its native plasma membrane. 相似文献
4.
Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. 总被引:46,自引:10,他引:36
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Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity. 相似文献
5.
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
6.
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
7.
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
8.
A novel gene fusion system has been constructed for fusions to the 5' end of gene zz, encoding a two-domain analogue of staphylococcal protein A designated ZZ. Four different genes were fused to the 5' end of zz, and their gene products were analyzed. One of the genes encodes a protein located intracellularly in Escherichia coli and the other three genes encode gene products destined for secretion across the cytoplasmic membrane by the presence of an amino terminal signal sequence. After production in E. coli, the fusion proteins were purified in a single step by IgG-affinity chromatography. The purified ZZ fusions could be used directly for amino terminal sequencing to confirm the start of translation of the intracellular product and the processing of the signal peptide of the translocated products. This is the first example of ZZ fusions to the C-terminus of gene products. To simplify the general use of fusions to the 5' end of zz, a new plasmid vector was constructed containing a multi restriction enzyme cloning linker and the lacZ' gene which enables screening for production in alpha-complementing supE strains of E. coli on indicator plates. 相似文献
9.
10.
Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1 beta 总被引:2,自引:0,他引:2
P Denèfle S Kovarik T Ciora N Gosselet J C Bénichou M Latta F Guinet A Ryter J F Mayaux 《Gene》1989,85(2):499-510
Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment. 相似文献
11.
The coding region for the mature form of TEM β–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 β–lactamase moiety had been translocated to the periplasm. Fusion proteins that contained ≤ 63 residues of PBP 1B possessed β–lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the 3–lactamase moiety of these fusion proteins remained in the cytoplasm. The β–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 trans–membrane segment (residues 64–87), with a short NH2–terminal domain (residues 1–63), and the remainder of the polypeptide (residues 68–844) 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
12.
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
13.
To reduce the number of deletion mutants from antibody (Ab) libraries that had been amplified by PCR from peripheral blood lymphocytes, we constructed the Ab expression vector, pLAB, in which DNA coding for a single-chain Ab was inserted into the gene encoding beta-lactamase (Bla) at the 3'-terminus of its signal sequence. After transforming Escherichia coli with this vector, a fusion protein with a functional Bla domain was produced that was able to protect the bacteria from the action of ampicillin (Ap). Libraries can therefore be usefully propagated with this vector, since only those clones carrying inserts that are in frame with Bla will survive Ap selection, while others that carry out-of-frame deletions or internal stop codons are eliminated. 相似文献
14.
Selection for signal sequence mutations that enhance production of secreted human proinsulin by Escherichia coli 总被引:1,自引:0,他引:1
This paper describes a method for the positive selection of signal sequence mutations that result in enhanced production of secreted human proinsulin by Escherichia coli. Coding sequences for the structural portion of beta-lactamase (EC 3.5.2.6) were substituted for those of the C terminus of proinsulin in a plasmid that normally directs the synthesis and secretion of proinsulin. The resulting plasmid directed the synthesis of a proinsulin/beta-lactamase fusion protein that was secreted into the periplasmic space and conferred resistance to low levels of ampicillin (Ap). Beneficial changes to the signal sequence were selected by the host's ability to grow on high levels of Ap. The beta-lactamase coding sequences were then replaced with those of human proinsulin, resulting in plasmids which directed enhanced production of secreted proinsulin. 相似文献
15.
The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins. 相似文献
16.
DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli. 相似文献
17.
High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein 总被引:2,自引:0,他引:2
A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily. 相似文献
18.
Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions. 总被引:5,自引:3,他引:2
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To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm. 相似文献
19.
20.
N V Iakubovich A G Zdanovski? A A Ianula?tis N K Iankovski? V G Debabov 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1987,(12):11-16
A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E. coli cells. The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene. The tox gene promoter is active in E. coli. The toxA protein is found mainly in periplasm of E. coli cells. The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells. An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid. The hybrid protein expresses both toxA and lacZ' activities. Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion. Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them. Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions. The existence of another translational start site functioning in E. coli and located inside 3'-end region of toxA mRNA is suggested. 相似文献