Recombinant Proteins In Bacteria Research Articles

Expression of Recombinant Cold-Adapted (Hemo)Globins from Marine Bacteria.

The production of recombinant proteins in bacteria made possible to obtain large quantities of proteins essential for basic and applied research. Escherichia coli remains one of the organisms of choice for recombinant proteins because of its ability to grow at high density and availability of a vast catalog of cloning vectors and mutant host strains. Here, we describe the protocols for the expression of cold-adapted (hemo)globins in Escherichia coli.

A combination strategy of solubility enhancers for effective production of soluble and bioactive human enterokinase

Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.

The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host\u2019s fluorescent intensity

BackgroundThe signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters.ResultsWhen fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain.ConclusionsWe identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression.

Structure-Function Relationship of Inclusion Bodies of a Multimeric Protein.

High level expression of recombinant proteins in bacteria often results in their aggregation into inclusion bodies. Formation of inclusion bodies poses a major bottleneck in high-throughput recovery of recombinant protein. These aggregates have amyloid-like nature and can retain biological activity. Here, effect of expression temperature on the quality of Escherichia coli asparaginase II (a tetrameric protein) inclusion bodies was evaluated. Asparaginase was expressed as inclusion bodies at different temperatures. Purified inclusion bodies were checked for biological activities and analyzed for structural properties in order to establish a structure-activity relationship. Presence of activity in inclusion bodies showed the existence of properly folded asparaginase tetramers. Expression temperature affected the properties of asparaginase inclusion bodies. Inclusion bodies expressed at higher temperatures were characterized by higher biological activity and less amyloid content as evident by Thioflavin T binding and Fourier Transform Infrared (FTIR) spectroscopy. Complex kinetics of proteinase K digestion of asparaginase inclusion bodies expressed at higher temperatures indicate higher extent of conformational heterogeneity in these aggregates.

A method of predicting the in vitro fibril formation propensity of A\u03b240 mutants based on their inclusion body levels in E. coli

Overexpression of recombinant proteins in bacteria may lead to their aggregation and deposition in inclusion bodies. Since the conformational properties of proteins in inclusion bodies exhibit many of the characteristics typical of amyloid fibrils. Based on these findings, we hypothesize that the rate at which proteins form amyloid fibrils may be predicted from their propensity to form inclusion bodies. To establish a method based on this concept, we first measured by SDS-PAGE and confocal microscopy the level of inclusion bodies in E. coli cells overexpressing the 40-residue amyloid-beta peptide, Aβ40, wild-type and 24 charge mutants. We then compared these results with a number of existing computational aggregation propensity predictors as well as the rates of aggregation measured in vitro for selected mutants. Our results show a strong correlation between the level of inclusion body formation and aggregation propensity, thus demonstrating the power of this approach and its value in identifying factors modulating aggregation kinetics.

A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins.

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.

Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail

The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments. Human HisGpn1 purity was higher than 95%, molecularly monodisperse and could be concentrated to more than 10 mg/mL without aggregating. Circular dichroism spectra showed that human HisGpn1 was properly folded and displayed a secondary structure rich in alpha helices. HisGpn1 effectively bound GDP and the non-hydrolyzable GTP analogue GMPPCP, and hydrolyzed GTP. We next tested the importance of the C-terminal tail, present in eukaryotic Gpn1 but not in the ancestral archaeal Gpn protein, on HisGpn1 dimer formation. C-terminal deleted human HisGpn1 (HisGpn1ΔC) was also purified as a protein dimer, indicating that the N-terminal GTPase domain contains the interaction surface needed for dimer formation. In contrast to HisGpn1, however, HisGpn1ΔC dimer spontaneously dissociated into monomers. In conclusion, we have developed a method to purify properly folded and functionally active human HisGpn1 from bacteria, and showed that the C-terminal tail, universally conserved in all eukaryotic Gpn1 orthologues, stabilizes the GTPase domain-mediated Gpn1 protein dimer. The availability of recombinant human Gpn1 will open new research avenues to unveil the molecular and pharmacological properties of this essential GTPase.

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

Recombinant expression, purification and osmotic regulation of in vitro activity of two enzymes constituting the Mozambique tilapia myo‐inositol biosynthesis pathway (1182.5)

To cope with hyperosmotic stress cells accumulate compatible organic osmolytes, which replace harmful inorganic electrolytes while maintaining homeostasis of cell volume and intracellular ionic strength. D‐myo‐Inositol (Ins) has been shown to be an important compatible organic osmolyte for fish that are exposed to salinity stress. Two enzymes are required to synthesize Ins: (I) MIPS (Ins phosphate synthase) converts glucose‐6‐phosphate to Ins‐3‐phosphate (Ins‐3‐P), and (II) IMPase (Ins monophosphate phosphatase) dephosphorylates Ins‐3‐P to yield Ins. Both enzymes are induced in different fish species and tissues after exposure to elevated environmental salinity, the result being intracellular accumulation of Ins. We cloned MIPS and IMPase from Mozambique tilapia (Oreochromis mossambicus), expressed the corresponding hexa‐His tagged recombinant proteins in bacteria, and purified recombinant MIPS and IMPase using Ni affinity chromatography. We show that both enzymes are more active at basic pH. In addition, the activity of these tilapia enzymes also depends on known cofactors for mammalian MIPS (NAD+) and IMPase (Mg2+). As is common for many other enzymes, tilapia MIPS activity decreases with increasing osmotic strength in vitro due to an increase in KM at elevated osmolality and a decrease of the kcat/KM ratio. In contrast, IMPase activity increases with elevated osmotic strength, particularly when Ins‐3‐P is used as a substrate. IMPase KM as well as Vmax and kcat all increase when buffer osmolality is elevated. These data suggest that, in addition to the induction of both enzymes at mRNA and protein levels, Ins accumulation during hyperosmotic stress is promoted by direct osmotic effects on IMPase enzymatic activity. Supported by NSF grant IOS‐1049780.

A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli

BackgroundProduction of recombinant proteins in bacteria for academic and commercial purposes is a well established field; however the outcomes of process developments for specific proteins are still often unpredictable. One reason is the limited understanding of the performance of expression cassettes relative to each other due to different genetic contexts. Here we report the results of a systematic study aiming at exclusively comparing commonly used regulator/promoter systems by standardizing the designs of the replicon backbones.ResultsThe vectors used in this study are based on either the RK2- or the pMB1- origin of replication and contain the regulator/promoter regions of XylS/Pm (wild-type), XylS/Pm ML1-17 (a Pm variant), LacI/PT7lac, LacI/Ptrc and AraC/PBAD to control expression of different proteins with various origins. Generally and not unexpected high expression levels correlate with high replicon copy number and the LacI/PT7lac system generates more transcript than all the four other cassettes. However, this transcriptional feature does not always lead to a correspondingly more efficient protein production, particularly if protein functionality is considered. In most cases the XylS/Pm ML1-17 and LacI/PT7lac systems gave rise to the highest amounts of functional protein production, and the XylS/Pm ML1-17 is the most flexible in the sense that it does not require any specific features of the host. The AraC/PBAD system is very good with respect to tightness, and a commonly used bioinformatics prediction tool (RBS calculator) suggested that it has the most translation-efficient UTR. Expression was also studied by flow cytometry in individual cells, and the results indicate that cell to cell heterogeneity is very relevant for understanding protein production at the population level.ConclusionsThe choice of expression system needs to be evaluated for each specific case, but we believe that the standardized vectors developed for this study can be used to more easily identify the nature of case-specific bottlenecks. By then taking into account the relevant characteristics of each expression cassette it will be easier to make the best choice with respect to the goal of achieving high levels of protein expression in functional or non-functional form.

N-Propanol Based Solubilization Buffer Enhances Refolding Yield of Inclusion Body Protein by Populating Intermediates to the Folding Pathway

Most of the times, high level expression of recombinant proteins in bacteria results in accumulation of recombinant proteins into inclusion body (IB) aggregates. To obtain native protein from these aggregates, it is necessary to solubilize these aggregates followed by refolding of solubilized protein by appropriate refolding method. Conventionally, high concentration of denaturant like urea or guanidinium chloride (GdmCl) is used for solubilization of inclusion bodies which often results into aggregation of protein during refolding process. In the present study we have evaluated a novel solubilization method using n-propanol in presence of low concentration of urea. n-Propanol based solubilization agent was compared with traditional solubilization agents like 8 M urea and 6 M GdmCl for solubilization efficiency, structure and stability of the solubilized model protein, recombinant human growth hormone (hGH). hGH IBs were found to be tough and were only solubilized efficiently in presence of high concentration of denaturants (8 M urea or 6 M GdmCl). 4 to 6 M n-propanol in combination with 2 M urea was sufficient for the efficient solubilization of hGH IBs. Aggregation during refolding was also studied and it was found that solubilization with n-propanol based buffer resulted into bioactive hGH without aggregation giving significantly higher refolding yield in comparison to those obtained with urea and GdmCl based buffers which resulted in aggregation of hGH during refolding. From the results obtained, it can be concluded that solubilization of hGH IBs in n-propanol based buffer results in a partially folded folding intermediate of hGH which readily folds into native form on dilution with reduced chances of protein getting into aggregation pathway.

Effects of a soy peptone on γ-IFN production steps in CHO-320 cells

High quality (glyco)proteins are needed for biopharmaceutical applications. They are generally expressed as recombinant proteins in bacteria, yeast, plant and/or animal cells in culture. Growth media are increasingly supplemented with peptones to improve either the cell growth or protein expression or both. To further enhance protein production, it is important to understand the effects of peptones at the cellular and molecular levels. Upon addition of a soy peptone to a cultivation medium of CHO cells, an increased specific productivity was observed. This work aimed at elucidating which main cellular functions are affected by the presence of the peptone. Therefore, hypotheses about putative effects on recombinant protein production steps have been postulated and tested. Although the effects emerge to be multiple, it was found that the peptone increased overall protein translation and recombinant protein secretion. Nonetheless, when other cellular functions (e.g. transcription, glycosylation, proteolytic degradation) were examined, no specific effects were observed. Further experiments are needed to probe the mechanisms related to the influence of the soy peptone on these cellular functions.

Strain engineering for improved expression of recombinant proteins in bacteria

Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins.

Production of Recombinant Proteins in Bacteria: The Inclusion Bodies Formation and their Use in Biomedicine

Four decades have passed since the first recombinant protein, somatostatin, was produced in Escherichia coli. To date, more than 160 biopharmaceuticals gained medical approval. As there is an increasing demand for new protein-based biopharmaceuticals and proteins used in diagnostics, novel or improved host organisms and expression systems are constantly being developed. Nevertheless, E. coli is still one of the most commonly used organisms for the production of recombinant therapeutic proteins. Despite many improvements of E. coli expression system, one major setback is protein aggregation and inclusion bodies (IBs) formation. These often cannot be prevented from forming during recombinant protein production. For a long time IB formation represented a problem in protein production. However, recent studies on bacterial cultivation and protein aggregation revealed that friendlier bacterial cultivation leads to production of non-classical IBs (ncIBs), which are composed of properly folded and biologically active proteins. Such active ncIBs can be used either for protein isolation (production) or as active nanoparticles. Since the field of nanobiotechnology is evolving fast, a further development of the use of active IBs is expected.

Regulation of Ephexin1, a Guanine Nucleotide Exchange Factor of Rho Family GTPases, by Fibroblast Growth Factor Receptor-mediated Tyrosine Phosphorylation

Fibroblast growth factor (FGF) signal is implicated in not only cell proliferation, but cell migration and morphological changes. Several different Rho family GTPases downstream of the Ras/ERK pathway are postulated to mediate the latter functions. However, none have been recognized to be directly coupled to FGF receptors (FGFRs). We have previously reported that EphA4 and FGFRs hetero-oligomerize through their cytoplasmic domains, trans-activate each other, and transduce a signal for cell proliferation through a docking protein, FRS2alpha (Yokote, H., Fujita, K., Jing, X., Sawada, T., Liang, S., Yao, L., Yan, X., Zhang, Y., Schlessinger, J., and Sakaguchi, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 18866-18871). Here, we have found that ephexin1, a guanine nucleotide exchange factor for Rho family GTPases, constitutes another downstream component of the receptor complex. Ephexin1 directly binds to the kinase domain of FGFR mainly through its DH and PH domains. The binding appears to become weaker and limited to the DH domain when FGFRs become activated. FGFR-mediated phosphorylation of ephexin1 enhances the guanine nucleotide exchange activity toward RhoA without affecting the activity to Rac1 or Cdc42. The FGFR-mediated tyrosine phosphorylation includes, but is not limited to, the residue (Tyr-87) phosphorylated by Src family kinase, which is known to be activated following EphA4 activation. The Tyr-to-Asp mutations that mimic the tyrosine phosphorylation in some of the putative FGFR-mediated phosphorylation sites increase the nucleotide exchange activity for RhoA without changing the activity for Rac1 or Cdc42. From these results, we conclude that ephexin1 is located immediately downstream of the EphA4-FGFR complex and the function is altered by the FGFR-mediated tyrosine phosphorylation at multiple sites.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

GST Pull-down

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. One of these applications is their use as probes for the identification of protein-protein interactions. The pull-down method described in this protocol is fundamentally similar to immunoprecipitation. Immunoprecipitation is based on the ability of an antibody to bind to its antigen in solution, and the subsequent purification of the immunocomplex by collection on protein A- or G-coupled beads. Similarly, the GST pull-down is an affinity capture of one or more proteins (either defined or unknown) in solution by its interaction with the GST fusion probe protein and subsequent isolation of the complex by collection of the interacting proteins through the binding of GST to glutathione-coupled beads.

Preparation of GST Fusion Proteins

INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.