Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization

Hirudin variant III (HV3) is potentially useful in the prevention and treatment of cataracts. To prepare sufficient amounts of rHV3 for further preclinical studies, we developed an effective process for robust preparative-scale extracellular production of rHV3 in Escherichia coli. In a 7-l bioreactor, under the optimal fed-batch fermentation conditions, rHV3 was excreted into the culture supernatant and yielded up to 915?mg?l^?1. Then, a four-step purification procedure was applied to the product, which included ultrafiltration, hydrophobic chromatography, anion-exchange chromatography, and preparative reversed-phase fast protein liquid chromatography (FPLC). The overall maximum recovery attained was 56?%, the purity reached at least 99?% as evaluated by HPLC analysis, the molecular weight was determined to be 7,011.10?Da by matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, and the pI was 4.46 as analyzed by isoelectric focusing. The N- and C-terminal sequence analysis confirmed the product homogeneity. The final product contained at most 10?pg?of residual DNA per dose (0.2?mg) of rHV3 by high-sensitivity hybridization assay and at most 3?EU?endotoxin protein/mg by limulus amebocyte lysate assay. Taken together, the rHV3 produced in multigram quantities in E. coli by this bioprocess meets the regulatory criteria for biopharmaceuticals and can be used as a drug candidate for preclinical studies.     

An improved strategy for high-level production of human vasostatin120-180

We previously reported a strategy for expression and purification of human Vasostatin120-180 (VAS), a potent angiogenesis inhibitor in a GST fusion form; however, the yield of 7.2 mg per liter of culture was relatively low. The aim of this study was to develop a more efficient system to improve and facilitate the production of VAS protein in a soluble and native form in Escherichia coli. The VAS gene with optimized condons was cloned into pET28a and overexpressed as a N-terminal His-tagged fusion protein. Between His-tag and VAS, an enterokinase recognition site was introduced to release the intact VAS. Optimal expression of soluble His-VAS was achieved by examining the contribution of chaperone coexpression and lower temperature fermentation. Ammonium sulfate precipitation was first employed to remove nucleic acid and partial host proteins. When further purified by Ni2+ affinity chromatography, 40 mg of His-VAS was isolated with purity over 85% from 1 L of culture. After desalting with Sephadex G15 and digestion with His-EK, followed by the removal of the His-tag and His-EK with Ni(2+)-NTA resin, 21 mg of intact VAS was finally obtained from 1 L of bacterial culture, which was approximately 3-fold the yield we previously obtained via GST fusion expression strategy. The identity of His-VAS and VAS was confirmed by Western blot. Purified VAS displayed distinct anti-angiogenic activity, which was shown by the endothelial cell proliferation inhibition assay and chicken chorioallantoic membrane assay. In sum, we greatly improved the yield of intact and bioactive VAS protein, and using this successful example, we propose a more efficient system for the high-level production of intact functional proteins, especially for low molecule weight peptides.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.     

An evolutionary strategy for isobutanol production strain development in Escherichia coli

Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σ^s-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production.     

An improved bulk purification method for Escherichia coli elongation factor, Ts

A bulk purification procedure has been designed to maximize the yield of Escherichia coli elongation factor, Ts, with a minimum of effort and time. The enzyme purification is achieved by DEAE-Sepharose and elongation factor Tu-affinity chromatographies. The typical yield is 150 mg/kg of E. coli (B) cells.     

dUTPase from Escherichia coli; high-level expression and one-step purification

The dut gene, which encodes Escherichia coli deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), has been recloned to increase overexpression of the enzyme and to enable simplification of the purification protocol into a one-step procedure. The gene was cloned into the vector pET-3a and expressed in E. coli BL21(DE3) pLysS under the control of a bacteriophage T7 promotor. Induction results in production of dUTPase corresponding to 60% of the extracted protein. Phosphocellulose chromatography at low pH was utilised for one-step purification, resulting in a homogenous preparation of the recombinant protein with a highly specific activity. The yield of purified enzyme is 500 mg per litre of bacterial culture, a significant increase compared to previously employed methods.     

An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli

DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.     

Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.     

An improved method for the purification of myrosinase and its physicochemical characterization

An improved high yielding procedure for the purification of myrosinase from Sinapis alba L. consisting of concanavalin A affinity chromatography followed by a chromatofocusing step is presented. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate-gel electrophoresis and by analytical ultracentrifugation although the presence of at least three isoenzymes, with pI values from 5.05 to 5.15, was detected by isoelectric focusing. It was found that the enzyme has a molecular weight of 135.1 kg mol-1 and consists of two, possibly identical, subunits of molecular weight 71.7 kg mol-1. The structure of myrosinase was studied by circular dichroism. Contin analysis of the CD data indicates a mixed alpha-helix and beta-sheet conformation for the native protein a with approximately 19% alpha-helix and approximately 35% beta-sheet content. Denaturation with guanidinium chloride was found to be irreversible although the enzyme has excellent storage characteristics in aqueous solution.     

In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT.

OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity Km of 0.4 microM for the substrate and a turnover number kcat of 40 s-1. The Km and kcat for the double variant were 1.1 microM and 1.6 s-1, respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.     

An Escherichia coli expression vector for high-level production of heterologous proteins in fusion with CMP-KDO synthetase

The construction of a vector which overproduces the enzyme, CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase or CKS) and its use as an expression vector for producing heterologous proteins in E. coli is described. The vector, which includes a modified lac promoter and synthetic ribosome binding site upstream of the native kdsB gene (encoding CKS), produces CKS at levels as high as 70% of the total cellular proteins. Several heterologous gene sequences have been fused to the 3'-end of the kdsB gene with resulting protein fusions produced at a level of up to 40% of the total cellular proteins.     

Biosensor-assisted evolution for high-level production of 4-hydroxyphenylacetic acid in Escherichia coli

4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, and biochemicals, and requires sustainable production to meet increasing demand. Here, we use a 4HPAA biosensor to overcome the difficulty of conventional library screening in identification of preferred mutants. Strains with higher 4HPAA production and tolerance are successfully obtained by atmospheric and room temperature plasma (ARTP) mutagenesis coupled with adaptive laboratory evolution using this biosensor. Genome shuffling integrates preferred properties in the strain GS-2-4, which produces 25.42 g/L 4HPAA. Chromosomal mutations of the strain GS-2-4 are identified by whole genome sequencing. Through comprehensive analysis and experimental validation, important genes, pathways and regulations are revealed. The best gene combination in inverse engineering, acrD-aroG, increases 4HPAA production of strain GS-2-4 by 37% further. These results emphasize precursor supply and stress resistance are keys to efficient 4HPAA biosynthesis. Our work shows the power of biosensor-assisted screening of mutants from libraries. The methods developed here can be easily adapted to construct cell factories for the production of other aromatic chemicals. Our work also provides many valuable target genes to build cell factories for efficient 4HPAA production in the future.     

Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.     

An improved method for the purification of Bacillus subtilis glucose dehydrogenase cloned in Escherichia coli

An improved and simplified purification procedure has been developed for the isolation of the Bacillus subtilis glucose dehydrogenase which has resulted in a 10 fold higher yield of pure enzyme. The purification procedure utilizes gene cloning and an additional ammonium sulfate step to facilitate the removal of contaminating proteins. The procedure requires fewer chromatographic steps than previously reported, thus simplifying the procedure. This improved and simplified purification of B. subtilis glucose dehydrogenase will facilitate further structure-function studies of this sporulation specific enzyme.     

Evaluation of Escherichia coli proteins that burden nonaffinity-based chromatography as a potential strategy for improved purification performance

Escherichia coli is a favored host for rapid, scalable expression of recombinant proteins for academic, commercial, or therapeutic use. To maximize its economic advantages, however, it must be coupled with robust downstream processes. Affinity chromatography methods are unrivaled in their selectivity, easily resolving target proteins from crude lysates, but they come with a significant cost. Reported in this study are preliminary efforts to integrate downstream separation with upstream host design by evaluating co-eluting host proteins that most severely burden two different nonaffinity-based column processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of green fluorescent protein (GFP) by hydrophobic interaction and anion exchange chromatography. Ribosomal protein L25 dominated non-target binding of polyarginine-tagged GFP on cation exchange resin. Implications for genetic knockout or site-directed mutagenesis resulting in diminished column retention are discussed for these and other identified contaminants.     

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L⁻¹ OD⁻¹. The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L^-1 OD⁻¹ of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3′-phosphoadenosine-5′-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.     

Optimization of BLyS production and purification from Escherichia coli

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.