Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in <Emphasis Type="Italic">Escherichia coli</Emphasis> using different carbon sources and chromatography sequences

Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.     

Prosequence switching: An effective strategy to produce biologically active E. coli heat-stable enterotoxin STh

Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.     

Obtaining of the p17 recombinant protein of human immunodeficiency subtype A virus

A simple and efficient method for expression in Escherichia coli cells and purification of a recombinant matrix protein, p17, of human immunodeficiency type I virus has been described. HIV-1 subtype A DNA sequence encoding p17 was obtained by amplification of the viral gag gene segment and cloned into an expression vector under the control of T7Lac promoter. The conditions for cell growth and induction of p17 synthesis by lactose and its further purification by metal chelate chromatography were optimized. p17 preparations with 97% purity were obtained; the yield of the protein of 28 mg per 1l of culture was achieved. The obtained protein was capable of binding antibodies from blood serum of a HIV-infected patient during immunoblotting.     

Ni2+-Based Immobilized Metal Ion Affinity Chromatography of Lactose Operon Repressor Protein from Escherichia Coli

Abstract

A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni²⁺-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.     

Bionanotechnology application of polypeptides in a hair color product: Self-assembly enables expression,processing, and functionality

Bionanotechnology aims to impart new properties to materials from unique functionalities present in biomolecules. However, the promise of bionanotechnology has not materialized beyond the biomedical field due in large part to issues of scalability, purity, and cost of manufacturing. In this work we demonstrate an approach to co-engineer production and system functionality into a single polypeptide. We designed a system to anchor particles onto hair via a multifunctional polypeptide composed of two domains, one with affinity to hair and the other capable of strong interactions with the particle surface. These strong interactions, exemplified by resistance to anionic surfactants, stem from the ability to self-assemble into higher order structures, which were observed by atomic force microscopy. At the same time, the controlled solubility properties of the particle binding domain permit the scalable production in Escherichia coli via inclusion bodies and cost effective purification. We believe this is a significant advance toward the development of bionanotechnology for industrial applications.     

An improved strategy for efficient expression and purification of soluble HIV-1 tat protein in <Emphasis Type="Italic">E.coli</Emphasis>

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.     

Predictive modeling of single pass tangential flow filtration for continuous biomanufacturing

Opportunities for process intensification have made continuous biomanufacturing an area of active research. While tangential flow filtration (TFF) is typically employed within the biologics purification train to increase drug substance concentration, single-pass TFF (SPTFF) modifies its format by enabling continuity of this process and achieving a multifold concentration factor through a single-pass over the filtration membranes. In continuous processes feed concentration and flow rate are determined by the preceding unit operations. Therefore, tight control of SPTFF output concentration must be achieved through precise design of the membrane configuration, unlike TFF. However, predictive modeling can be utilized to identify configurations that achieve a desired target concentration across ranges of possible feed conditions with minimal experimental data, hence enabling accelerated process development and design flexibility. We hereby describe the development of a mechanistic model predicting SPTFF performance across a wide design space using the well-established stagnant film model, which we demonstrate is more accurate at higher feed flow rates. The flux excursion dataset was generated within time constraints and with minimal material consumption, showing the method's ability to be quickly adapted. While this approach eliminates characterizing complex physicochemical model variables or the need for users with specialized training, the model and its assumptions become inaccurate at low flow rates, below 25 L/m²/h, and high conversions, above 0.9. As this low flow rate, high conversion operating regime is relevant for continuous biomanufacturing, we explore the assumptions and challenges involved in predicting and modeling SPTFF processes, while suggesting added characterization to gain further process insight.     

Overexpression and purification of the galactose operon enzymes from Escherichia coli

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically cheracterized by gel-filtration column chromatography and isoelectric focusing, and the K_m values for their substrates were determined.     

Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

A self‐cleaving elastin‐like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self‐cleaving ELP‐intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP‐intein purification. Thus, this simple ELP‐based method should be applicable for the nonchromatographic purification of other recombinant, in vivo‐assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large‐scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.     

Simple purification ofEscherichia coli-derived recombinant human interleukin-2 expressed with N-terminus fusion of glucagon

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed inEscherichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced inE. coli cytoplasm were easily dissolved by simple alkaline pH shift (8→12→8). Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.     

Flexprep: Scale-flexible rapid plasmid preparation for analysis of recombinant clones

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones. this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3–10 kbp plasmid vectors in <10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.     

Thymol-triggered lysis of Escherichia coli expressing Lactobacillus phage LL-H muramidase

Effective disruption of Escherichia coli cells is achieved by the intracellularly accumulated recombinant murein hydrolase (Lactobacillus bacteriophage LL-H muramidase) after the addition of 5 mM thymol. Thymol destroys the integrity and electric potential of the cytoplasmic membrane, and as a consequence the muramidase can access and hydrolyze the cell wall murein leading to cell lysis. Lysis occurred within 5 min after the addition of thymol and seemed to be efficient at high culture densities. This lysis method does not require cell harvesting or addition of other cell wall weakening substances or exogenous enzymes. As a cell disruption method, thymol-triggered lysis is as efficient as sonication in the presence of 1% Triton. Furthermore, thymol did not interfere with the purification steps of Mur by expanded bed adsorption chromatography (EBA), suggesting that the lysis method presented here is well suited for large-scale production and purification of intracellular proteins of E. coli. Received 21 April 1998/ Accepted in revised form 5 December 1998     

A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli

Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC₅₀ was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli.

     

Addition of a surfactant to tryptic soy broth allows growth of a lactic acid bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica

Aims: This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture. Methods and Results: Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l^?1 Tween 80^® (TSB‐T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB‐T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB‐T80 but was significantly less in TSB. Conclusions: Supplementation of TSB with Tween 80^® allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro. Significance and Impact of the Study: This study reports the utility of TSB‐T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.     

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (k_ox), responses to physiological conditions (pH, [Cl^?], [IHP], and T), and ligand binding kinetics (O₂, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O₂ carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.     

Efficient method for preparation of highly purified lipopolysaccharides by hydrophobic interaction chromatography

A method for the efficient preparation of highly purified lipopolysaccharides (LPSs) by hydrophobic interaction chromatography (HIC) has been developed. The procedure can be used for the purification of cell wall bound LPSs after hot phenol–water extraction and for the isolation of extracellular LPSs from the supernatant, respectively. The method described has been tested with artificial mixtures containing LPSs, polysaccharide, protein and RNA and subsequently employed for the preparative purification of two LPSs of different origin, namely the extracellular LPS secreted by Escherichia coli E49 into the culture medium, and the cell wall bound LPS from Pseudomonas aeruginosa VA11465/1. Compared to currently used methods for LPS purification such as enzymatic digestion and ultracentrifugation, the chromatographic separation reported here combines superior purity with minimal loss of LPS, high reproducibility and simple handling. The removal of contaminants such as protein, RNA and polysaccharides and the recovery of LPSs were monitored by appropriate assays.     

Development of a novel purification method for AAV vectors using tangential flow filtration

Adeno-associated virus (AAV) vector can efficiently transduce therapeutic genes in various tissue types with less side effects; however, owing to complex multistep processes during manufacture, there have been surges in the pricing of recently approved AAV vector-based gene therapy products. This study aimed to develop a simple and efficient method for high-quality purification of AAV vector via tangential flow filtration (TFF), which is commonly used for concentration and diafiltration of solutions during AAV vector purification. We established a novel purification method using TFF and surfactants. Treatment with two classes of surfactants (anionic and zwitterionic) successfully inhibited the aggregation of residual proteins separated from the AAV vector in the crude product by TFF, obtaining a clearance of 99.5% residual proteins. Infectivity of the AAV vector purified using the new method was confirmed both in vitro and in vivo, and no remarkable inflammation or tissue damage was observed in mouse skeletal muscle after local administration. Overall, our proposed method could be used to establish a platform for the purification of AAV vector.     

Lactic acid bacteria biofilms and their ability to mitigate Escherichia coli O157:H7 surface colonization

Lactic acid bacteria (LAB) exert antagonistic activities against diverse microorganisms, including pathogens. In this work, we aimed to investigate the ability of LAB strains isolated from food to produce biofilms and to inhibit growth and surface colonization of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 at 10°C. The ability of 100 isolated LAB to inhibit EHEC O157:H7 NCTC12900 growth was evaluated in agar diffusion assays. Thirty-seven LAB strains showed strong growth inhibitory effect on EHEC. The highest inhibitory activities corresponded to LAB strains belonging to Lactiplantibacillus plantarum, Pediococcus acidilactici and Pediococcus pentosaceus species. Eighteen out of the 37 strains that showed growth inhibitory effects on EHEC also had the ability to form biofilms on polystyrene surfaces at 10°C and 30°C. Pre-established biofilms on polystyrene of four of these LAB strains were able to reduce significantly surface colonization by EHEC at low temperature (10°C). Among these four strains, Lact. plantarum CRL 1075 not only inhibited EHEC but also was able to grow in the presence of the enteric pathogen. Therefore, this strain proved to be a good candidate for further technological studies oriented to its application in food-processing environments to mitigate undesirable surface contaminations of E. coli.     

Expression, Renaturation and Simultaneous Purification of Recombinant Human Stem Cell Factor in Escherichia coli

Recombinant human stem cell factor (rhSCF) was produced as an inclusion body by Escherichia coli DH5α grown in a 5 l fermentor. Inclusion bodies of rhSCF were purified and solubilized in urea solution, then renatured with simultaneous purification using a high performance hydrophobic interaction chromatographic (HPHIC) squat column. The refolded rhSCF had a purity of 94% and a bioactivity of 1.2 × 10⁶ IU mg⁻¹of rhSCF protein. The method described is fast and simple to implement.