Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography

Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion‐exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10⁸ L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:864–871, 2014     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.     

Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

NP_Δc375 is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self‐assembles into a long helical structure. A packed bed anion exchange chromatography (PB‐AEC), SepFastTM Supor Q pre‐packed column, was used to purify NP_Δc375 from clarified feedstock. This PB‐AEC column adsorbed 76.2% of NP_Δc375 from the clarified feedstock. About 67.5% of the adsorbed NP_Δc375 was successfully eluted from the column by applying 50 mM Tris‐HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB‐AEC operation. Electron microscopic analysis revealed that the helical structure of the NP_Δc375 purified by SepFast^TM Supor Q pre‐packed column was as long as 490 nm and 22–24 nm in diameter. The antigenicity of the purified NP_Δc375 was confirmed by enzyme‐linked immunosorbent assay. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 564–567, 2013     

Expression and characterization of recombinant human cytochrome c in E. coli

Cytochrome c is a heme protein involved in electron transfer, cell apoptosis, and diseases associated with oxidative stress. Here we expressed human cytochrome c in E. coli and purified it to homogeneity with a yield of 10–15 mg/L. The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic voltammetry. This is similar to that of horse cytochrome c with a value of 0.249 V. The sequential assignment and structural analysis of recombinant human ferrocytochrome c were obtained using multidimensional NMR spectroscopy. On the basis of our NMR studies, the recombinant human cytochrome c produced in E. coli exhibits the same tertiary fold as horse cytochrome c. These results provide evidence that human cytochrome c expressed in E. coli possesses a similar function and structure to that of the horse protein. It is known that cytochrome c plays a role in many human diseases. This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.     

Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.     

Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange

The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.     

Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli

The HSPA6, one of the members of large family of HSP70, is significantly up-regulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant camel HSPA6 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of cHSPA6 was increased proportionally with increased incubation temperature up to 37 °C. Induction with 10 μM IPTG was sufficient to induce the expression of cHSPA6 which was 100 times less than normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of cHSPA6 in soluble form. In addition, 5 h of post-induction incubation was found to be optimal to produce folded cHSPA6 with higher specific and volumetric yield. Subsequently, highly pure and homogenous cHSPA6 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant HSPA6 protein from Camelus dromedarius in Escherichia coli in milligram quantities from shake flask liquid culture.     

Pro-sequence and Ca2+-binding: implications for folding and maturation of Ntn-hydrolase penicillin amidase from E. coli

Penicillin amidase (PA) is a bacterial periplasmic enzyme synthesized as a pre-pro-PA precursor. The pre-sequence mediates membrane translocation. The intramolecular pro-sequence is expressed along with the A and B chains but is rapidly removed in an autocatalytic manner. In extensive studies we show here that the pro-peptide is required for the correct folding of PA. Pro-PA and PA unfold via a biphasic transition that is more pronounced in the case of PA. According to size-exclusion chromatography and limited proteolysis experiments, the inflection observed in the equilibrium unfolding curves corresponds to an intermediate in which the N-terminal domain (A-chain) still possesses native-like topology, whereas the B-chain is unfolded to a large extent. In a series of in vitro experiments with a slow processing mutant pro-PA, we show that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude. In the absence of the pro-domain the PA refolds to a stable inactive molten globule intermediate that has native-like secondary but little tertiary structure. The pro-sequence of the homologous Alcaligenes faecalis PA can facilitate the folding of the hydrolase domain of Escherichia coli PA when added in trans (as a separate polypeptide chain). The isolated pro-sequence has a random structure in solution. However, difference circular dichroism spectra of native PA and native PA with pro-peptide added in trans suggest that the pro-sequence adopts an alpha-helical conformation in the context of the mature PA molecule. Furthermore, our results establish that Ca2+, found in the crystal structure, is not directly involved in the folding process. The cation shifts the equilibrium towards the native state and facilitates the autocatalytic processing of the pro-peptide.     

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.     

Arabinogalactan protein cluster from Jatropha curcas seed embryo contains fasciclin,xylogen and LysM proteins

An non-GPI-anchored AGP cluster (Y2) was isolated from the seeds of Jatropha curcas L. (Euphorbiaceae) composed of 4.8% polypeptides (mainly Ala, Ser, Gly, Hyp, Glu) and a carbohydrate moiety composed of Gal, Ara, GlcA, Rha, Man and GlcN. Besides the typical structural features of arabinogalactan proteins, typical N-glycan linker of the complex type (GlcNAc₄Man₃Gal₂Fuc₁Xyl₁) were identified. O-glycosylation occurred mainly via Hyp and to a lesser extent via Thr and Ser. N-glycans from the complex type, carrying at the innermost GlcNAc at position O-3 one α-Fuc-residue, were also present.     

Analytical biotechnology of recombinant peptides and proteins: II. A confirmation of the primary structure of fusion protein containing human proinsulin and optimization of its proteolysis by trypsin

The kinetics of trypsin proteolysis of the fusion protein (FP) containing human proinsulin was studied by a set of analytical micromethods. These were the microcolumn reversed-phase HPLC and the qualitative identification by MALDI-TOF mass spectrometry and amino acid sequencing. The first stage of the proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off ofC-peptide from proinsulin results in the formation of Arg^B31-Arg^B32-insulin. The effect of temperature on the formation of de-Thr^B30-insulin, a by-product, was also studied. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain noN-terminal Met residue. For communication I, see [1].     

Cloning and expression of soluble recombinant protein comprising the extracellular domain of the human type I interferon receptor 2c subunit (IFNAR-2c) in E. coli

An optimized procedure was developed for production of the extracellular domain encoding amino acids 1–243 of the human type I interferon receptor 2c subunit (IFNAR-2c) as a fusion protein with glutathione S-transferase (GST-IFNAR2cEC) in E. coli to obtain active, soluble protein. Induction of protein expression at 37 °C resulted in a formation of inclusion body. Co-expression with bacterial chaperones, GroEL and GroES, failed to support the folding of GST-IFNAR2cEc under IPTG induction at 37 °C. Expression induced at 25 °C decreased the inclusion body formation up to 62% and cell disruption by a French press provided higher recovery of the recombinant protein than cell disruption by sonication.     

A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli

Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His₆-tag and Trx-His₆-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His₆-hLIF fusion protein was not as soluble as the Trx-His₆-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (>95% pure) His₆-hLIF and Trx-His₆-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His₆-hLIF and Trx-His₆-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method.     

A combination of cation exchange and ligand-affinity chromatography for purification of two molecular species of the folate binding protein in human milk,one equipped with a hydrophobic glycosyl phosphatidylinositol tail: characterization of hydrophobicity and electrical charge

Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5-6 was a cleavage product of a 100 kDa protein eluted at pH 3-4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7-9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.     

Methionyl-tRNA synthetase from E. coli: direct evidence for exchange of protomers in the dimeric enzyme by using deuteration and small-angle neutron scattering

Direct demonstration of the reversible dissociation of native dimeric methionyl-tRNA synthetase from E. coli has been obtained using small angle neutron scattering and deuterated enzyme. Structural parameters of the fully deuterated dimer are very similar to the hydrogenated one. Analysis of the variations of the intensity and of the radius of gyration of a stoichiometric mixture of the two types of dimer (hydrogenated and deuterated), as a function of D2O content in the solvent, enabled us to characterize an hybrid dimer, having both hydrogenated and deuterated protomers. By separating the contribution of each protomer to the scattering, the radius of gyration of the protomer in situ and the distance between the centers of mass of each protomer in the dimer are determined.     

Identification of recombinant AtPYL2, an abscisic acid receptor,in E. coli using a substrate-derived bioactive small molecule,a biotin linker with alkyne and amino groups,and a protein cross-linker

Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3′-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).     

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.