Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli

Significant leakage of host-cell proteins into the culture medium occurred during high-cell-density cultivation of E. coli. Identification of these medium proteins revealed almost exclusively a periplasmic origin. Release of periplasmic proteins into the culture medium was observed throughout the entire cultivation of recombinant or non-recombinant cells. Leakage was intensified, however, in the final part of high-cell-density cultures (>100 g L(-)(1) dry cell mass) or when a temperature upshift was used for induction of recombinant protein synthesis. After temperature upshift, formation rates and residual cellular concentrations of periplasmic proteins declined with individual rates; e.g., the cellular content of the large periplasmic dipeptide binding protein DppA (57.4 kDa) started to decline about 4 h after the temperature upshift, whereas the smaller periplasmic d-galactose/d-glucose binding protein MglB (33.4 kDa) was already lost during the first hour after the upshift. In addition to periplasmic proteins, the osmotic-shock-sensitive heat-shock protein DnaK was found in significantly higher proportion in the cell-free medium of the temperature-challenged culture than other cytoplasmic proteins. Cell lysis was not observed even after prolonged cultivation. Thus, loss of a subset of cellular proteins of mainly periplasmic origin ordinarily occurs during cultivation and is intensified through stressful conditions in high-cell-density cultures. The selective release of cellular proteins of periplasmic origin offers the opportunity to simplify the downstream processing of recombinant proteins directed to the periplasm of E. coli.     

大肠杆菌周质蛋白提取工艺的研究

研究了精氨酸缓冲液在不同浓度、pH值及提取时间下的周质蛋白提取率,并以溶菌酶法、渗透压休克法作为对比。结果表明浓度0.4 mol/L,pH值8.0,提取时间为45 min时,周质目的蛋白达到0.89 mg/g湿菌,相比其他方法,周质蛋白提取率分别提高93%、187%。实验得到一种高效、方便的大肠杆菌周质蛋白提取工艺,为周质表达的重组蛋白大规模生产奠定了基础。     

Expression in periplasmic space of Shewanella oneidensis

A Shewanella expression system has been used for an overproduction of c-type multiheme proteins. The proteins were exported to the periplasmic space for the maturation. Since the periplasmic expression system is attractive, especially for protease-sensitive proteins, an expression vector containing a signal peptide was constructed for expressions in the periplasmic space of Shewanella oneidensis. To evaluate the system, two eukaryotic proteins which originally do not have signal sequences and are difficult to express in Escherichia coli, were selected. The first is human cytochrome c. Properties of the recombinant cytochrome c were identical to those previously reported, indicating the protein is intact. The other was potato calcium-dependent protein kinase. The protein was expressed in periplasmic space. These results indicated that the system is generally applicable for any protein expression including c-type cytochromes, protease-sensitive proteins and those with multi-disulfide bonds because of transportation to the periplasmic space.     

A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.     

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.     

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.     

Two cysteines in each periplasmic domain of the membrane protein DsbB are required for its function in protein disulfide bond formation.

DsbB is a protein component of the pathway that leads to disulfide bond formation in periplasmic proteins of Escherichia coli. Previous studies have led to the hypothesis that DsbB oxidizes the periplasmic protein DsbA, which in turn oxidizes the cysteines in other periplasmic proteins to make disulfide bonds. Gene fusion approaches were used to show that (i) DsbB is a membrane protein which spans the membrane four times and (ii) both the N- and C-termini of the protein are in the cytoplasm. Mutational analysis shows that of the six cysteines in DsbB, four are necessary for proper DsbB function in vivo. Each of the periplasmic domains of the protein has two essential cysteines. The two cysteines in the first periplasmic domain are in a Cys-X-Y-Cys configuration that is characteristic of the active site of other proteins involved in disulfide bond formation, including DsbA and protein disulfide isomerase.     

SurA assists the folding of Escherichia coli outer membrane proteins.

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.     

Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli

Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds. In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway. Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site. Most of them have a domain that displays the thioredoxin-like fold. In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity. Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors. This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E. coli.     

Periplasmic competition for zinc uptake between the metallochaperone ZnuA and Cu,Zn superoxide dismutase

We have investigated the availability of zinc in the periplasmic space of Escherichia coli using a mutant Cu,Zn superoxide dismutase whose dimerization is triggered by zinc binding. This mutant enzyme accumulates in the monomeric form when wild type cells are grown in minimal medium, but assembles in the dimeric form when it is produced in the same medium by a mutant strain lacking the periplasmic zinc metallochaperone ZnuA. These results indicate that periplasmic zinc-containing proteins compete for metal binding when bacteria grow in environments where this element is present in traces. The effective ZnuA ability to sequester the available zinc ions from the periplasm suggests that zinc-containing cytoplasmic proteins are more important for bacterial viability than the periplasmic ones.     

大肠杆菌周质蛋白提取工艺的改进

介绍一种简捷高效的大肠杆菌周质蛋白提取工艺,即用含一定浓度溶菌酶的细胞裂解缓冲液一步提取周质蛋白,与传统的高渗和低渗两步提取法相比,不仅操作简单快捷,并且显著的提高了大肠杆菌周质蛋白的提取率.     

Periplasmic proteins of Escherichia coli are highly resistant to aggregation: reappraisal for roles of molecular chaperones in periplasm

Periplasmic proteins of Gram-negative bacteria like Escherichia coli are subjected to immediate affect of environmental fluctuation that may unfold proteins, due to the permeability of the outer membrane to small molecules. They are thus supposedly protected by certain molecular chaperones. Nevertheless, no homologues of typical molecular chaperones have so far been found in periplasm, and the recently reported chaperone activities of periplasmic protein disulfide isomerase (PDI) and peptidyl prolyl isomerase (PPI) seem to be too weak to satisfy such assumed needs. In an attempt to reveal whether periplasmic proteins exhibit certain unusual properties, we discovered that such proteins as a whole are highly resistant to aggregation under a wide variety of denaturing conditions. Furthermore, in an effort to unveil the nature behind this phenomenon we purified and examined four prominent periplasmic proteins. Our results demonstrate that these proteins unfold at rather mild denaturing conditions and expose hydrophobic surfaces during such unfolding process, but hardly form complexes with a typical molecular chaperone. Based on these observations, we propose that the periplasmic proteins have been evolved to resist the formation of aggregates when subjected to various denaturing conditions and molecular chaperones may thus not be needed in periplasm.     

Simple, rapid, and quantitative release of periplasmic proteins by chloroform.

We introduce a method by which periplasmic proteins can be released rapidly, simply, and quantitatively by treating cells with chloroform. All the amino acid-binding proteins tested maintained their activity during chloroform treatment. This method makes practical the analysis of the periplasmic protein complement of a large number of strains.     

Two Different Types of Dehalogenases, LinA and LinB, Involved in γ-Hexachlorocyclohexane Degradation in Sphingomonas paucimobilis UT26 Are Localized in the Periplasmic Space without Molecular Processing

gamma-Hexachlorocyclohexane (gamma-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems. The cellular proteins of a gamma-HCH-degrading bacterium, Sphingomonas paucimobilis UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock. Most of two different types of dehalogenase, LinA (gamma-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of gamma-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing. Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins. LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism.     

Extraction of recombinant periplasmic proteins under industrially relevant process conditions: Selectivity and yield strongly depend on protein titer and methodology

In this work, we attempted to identify a method for the selective extraction of periplasmic endogenously expressed proteins, which is applicable at an industrial scale. For this purpose, we used an expression model that allows coexpression of two fluorescent proteins, each of which is specifically targeted to either the cytoplasm or periplasm. We assessed a number of scalable lysis methods (high-pressure homogenization, osmotic shock procedures, extraction with ethylenediaminetetraacetic acid, and extraction with deoxycholate) for the ability to selectively extract periplasmic proteins rather than cytoplasmic proteins. Our main conclusion was that although we identified industrially scalable lysis conditions that significantly increased the starting purity for further purification, none of the tested conditions were selective for periplasmic protein over cytoplasmic protein. Furthermore, we demonstrated that efficient extraction of the expressed recombinant proteins was largely dependent on the overall protein concentration in the cell.     

AMIN domains have a predicted role in localization of diverse periplasmic protein complexes

We describe AMIN (Amidase N-terminal domain), a novel protein domain found specifically in bacterial periplasmic proteins. AMIN domains are widely distributed among peptidoglycan hydrolases and transporter protein families. Based on experimental data, contextual information and phyletic profiles, we suggest that AMIN domains mediate the targeting of periplasmic or extracellular proteins to specific regions of the bacterial envelope.     

The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.     

The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.

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.     

Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease. Comparison with other members of the family.

The membrane-bound complex of periplasmic permeases comprises two hydrophobic proteins which have been hypothesized to be integral membrane-spaninning proteins. We have investigated the topological organization of the hydrophobic components of the Salmonella typhimurium histidine permease, HisQ and HisM. Both proteins are digested by trypsin and proteinase K when either inside-out or right-side-out membrane vesicles are used. Therefore, these proteins are exposed to both surfaces of the membrane. Digestion with carboxypeptidase and binding studies with antibodies directed against the carboxyl terminus of HisQ and HisM have localized their carboxyl termini to the inside surface of the cytoplasmic membrane. Aminopeptidase digestion suggests periplasmic localization of their amino termini. Alkaline phosphatase fusions to HisQ and HisM indicate the existence of five spanners in both proteins. The periodicity and orientation of spanners and loops in HisQ and HisM match those of the five carboxyl-terminal spanners of MalF, the only other hydrophobic component of the periplasmic permeases for which topological information is available. An alignment of the sequences of all known hydrophobic components of periplasmic permeases is presented which indicates clear conservation of secondary structure and some conservation of primary sequence. The structural conservation of the components is discussed, and a role for a hydrophilic loop containing a conserved sequence (the EAA loop) is proposed.