Expression of aChlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein

A single-chain antibody fragment has been constructed for an antibody that binds to theChlamydia specific carbohydrate structure of the lipopolysaccharide. Single-chain protein was expressed and secreted into the periplasmic space ofE. coli as a fusion protein with the maltose binding protein. The fusion protein was purified in one step by virtue of its ability to bind to maltose. In a sandwich ELISA, the eluted protein boundChlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein. The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.     

Secretion of recombinant proteins from E. coli

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.     

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (q_S) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and q_S are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g⁻¹, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

Extracellular recombinant protein production from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.     

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

DEVELOPMENT OF GENETICALLY ENCODED FLUORESCENT PROTEIN CONSTRUCTS OF HYPERTHERMOPHILIC MALTOSE-BINDING PROTEIN

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

Eukaryotic integral membrane protein expression utilizing the <Emphasis Type="Italic">Escherichia coli</Emphasis> glycerol-conducting channel protein (GlpF)

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).     

Factors that influence the extracellular expression of streptavidin in <Emphasis Type="Italic">Escherichia coli</Emphasis> using a bacteriocin release protein

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.     

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.     

Genomic cloning,expression and recombinant protein purification of α and β subunits of the allophycocyanin gene <Emphasis Type="Italic">(apc)</Emphasis> from the cyanobacterium <Emphasis Type="BoldItalic">Anacystis nidulans</Emphasis> UTEX 625

A genomic fragment encoding α^APC and β^APC (i.e., α and β units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP (maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. α^APC and β^APC were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications.     

The soluble expression of the human renin binding protein using fusion partners: A comparison of ubiquitin,thioredoxin, maltose binding protein and NusA

human renin binding protein (hRnBp), showingN-acetylglucosamine-2-epimerase activity, was over-expressed inE. coli, but was mainly present as an inclusion body. To improve its solubility and activity, ubiquitin (Ub), thioredoxin (Trx), maltose binding protein (MBP) and NusA, were used as fusion partners. The comparative solubilities of the fusion proteins were, from most to least soluble: NusA, MBP, Trx, Ub. Only the MBP fusion did not significantly reduce the activity of hRnBp, but enhanced the stability. The Origami (DE3), permitting a more oxidative environment for the cytoplasm inE. coli, helped to increase its functional activity.     

Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system

The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.     

Two distinct regions in the model protein Peb1 are critical for its heterologous transport out of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host.     

Secretory and extracellular production of recombinant proteins using<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.     

A Pair of Ligation-Independent Escherichia coli Expression Vectors for Rapid Addition of a Polyhistidine Affinity Tag to the N- or C-Termini of Recombinant Proteins

6×His tag is one of the most widely used affinity fusion tags that facilitates detection and purification of recombinant proteins. However, the location of this tag within a particular type of protein may influence the expression, solubility, and bioactivity of the protein, and the optimal location needs to be determined experimentally. To provide a tool for rapid generation of 6× His tags at the N- or C-terminus of any recombinant protein, we have constructed a pair of Escherichia coli expression vectors—pLIC-NHis and pLIC-CHis—based on the pET30a vector, for ligation-independent cloning (LIC). Construction of this new pair of LIC vectors was accomplished by replacement of the multiple cloning site of pET30a with two specifically designed LIC cloning sites. A target gene derived by PCR with a pair of predesigned primers can be inserted into the LIC site of pLIC-NHis for expression of recombinant proteins fused with the N-terminal sequence MHHHHHHG or into that of pLIC-CHis for expression of recombinant proteins with the C-terminal sequence THHHHHH. Successful expression of two normal mammalian prion proteins and five bacterial proteins in E. coli using this pair of LIC vectors reveals that these vectors are valuable tools for the production of recombinant His-tagged proteins in E. coli.     

Serological Detection of Grapevine leafroll virus 2 Using an Antiserum Developed against the Recombinant Coat Protein†

Grapevine leafroll associated virus 2 (GLRaV 2) is one of the important components in the leafroll disease complex. The coat protein gene of GLRaV 2 was cloned into a protein expression vector pMAL‐c2x and the recombinant protein, consisting of the maltose binding protein (MBP) and GLRaV 2 coat protein (CP), was expressed in Escherichia coli. The recombinant MBP‐CP was used to raise a high quality antiserum. When used in Western blot analysis, the anti‐MBP‐CP antiserum produced specific reaction to the recombinant protein as well as to the viral coat protein of GLRaV 2. In Immunosorbent electron microscopy study, the anti‐MBP‐CP antibodies strongly decorated the GLRaV 2 virions. Using the newly developed antiserum, an indirect plate‐trapped antigen enzyme‐linked immunosorbent assay method was developed and successfully implemented for virus detection. A field survey was conducted to evaluate the virus infection status by GLRaV 2 and GLRaV 3 using antibodies developed against their respective recombinant coat proteins.