Mohammad M. Ataai

Selection of optimum affinity tags from a phage-displayed peptide library. Application to immobilized copper(II) affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a versatile tool for the purification of proteins with affinity for immobilized metals. Moreover, this technique has also been used for the separation of proteins that do not exhibit significant metal affinity in the native form, by their fusion to a short metal-binding peptide (a tail), most commonly, a sequence consisting of six adjacent histidine residues (His6). A phage-displayed random hexamer library is used to select for peptides with affinity for immobilized copper. The study follows our previous investigation in which a stringent selection protocol led to the selection of only one copper-binding peptide containing two histidines. The less stringent conditions employed in this work resulted in the selection of a more diverse population of peptides, but again, dominated by peptides containing two histidines (13 out of 19). The prevalence of peptides with two histidines, in contrast to peptides with a higher number of histidines (e.g. His6 or HHHMVH), is explained based on the differences in the pH dependence of their affinity for copper. As discussed, the selected peptides with two histidines will be superior affinity tails than peptides with a higher histidine content (e.g. His6). Moreover, a peptide with a single histidine but with a very high copper affinity, is also identified. Its high copper affinity is related to the presence of several hydrophobic residues in the neighborhood of histidine. Chromatography of human interleukin-1 beta (hIL-1 beta) and several other proteins containing a single surface-exposed histidine surrounded by several hydrophobic residues confirmed that such a sequence could also serve as a very effective metal binding domain for protein purification using immobilized copper(II) columns.

Characterization of Growth and Acid Formation in a Bacillus subtilis Pyruvate Kinase Mutant

ABSTRACT Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pykmutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of thepyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO2 production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pykmutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37 degrees C. At 42 degrees C, its plasmid content was ninefold higher than JM101 at 37 degrees C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp(uv), under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

BackgroundPlasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production.ResultsSuch a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmids antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date.ConclusionThese results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that reduce acetate production would also be advantageous. The results further suggest that using some other means for plasmid selection than antibiotic resistance, or at least weakening the markers expression, would be beneficial because it would allow more precursor metabolites, energy, and reducing power to be put toward plasmid production. Thus far, the impact of eliminating Pyk activity has been explored experimentally, with significantly higher plasmid yields resulting.

Engineering of Bacillus subtilis for Enhanced Total Synthesis of Folic Acid

ABSTRACT We investigated whether the yield of the B vitamin folic acid could be elevated in Bacillus subtilis. Strategies for increasing the folic acid yield were investigated by employing computer-aided flux analysis and mutation. Controlling the activity of the enzyme pyruvate kinase by placing it under inducible control was one strategy devised to elevate yield while insuring that a rapid growth rate results. Other single mutation strategies included amplifying the expression of the genes in the folate operon and overexpressing the Escherichia coli aroH gene, which encodes 2-dehydro-3-deoxyphosphoheptonate aldolase. The latter could conceivably elevate the abundance of the folic acid precursor, para-aminobenzoic acid. Strains that combined two or more mutations were also constructed. Overall, a strain possessing inducible pyruvate kinase, overexpressed aroH, and increased transcription and translation of genes from the folic operon exhibited the best yield. The yield was eightfold higher than that displayed by the parent B. subtilis 168 strain.

Cell Growth and By‐Product Formation in a Pyruvate Kinase Mutant of E. coli

In this paper, we report on the analysis of acid formation in an E. coli pyk mutant. The results demonstrate that acid formation is insignificant for both the wild‐type and the mutant at low glucose concentrations. However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild‐type. This substantial reduction in acids is accompanied by an increase in CO2 production. Moreover, unlike the B. subtilispyk mutant, the E. coli pyk mutant did not show a substantial increase in the PEP pool.

Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification

ABSTRACT Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications, particularly at peripheral nerves, the natural site of virus latency. Many gene vectors require large particle numbers for even early-phase clinical trials, emphasizing the need for high-yield, scalable manufacturing processes that result in virus preparations that are nearly free of cellular DNA and protein contaminants. HSV-1 is an enveloped virus that requires the development of gentle purification methods. Ideally, such methods should avoid centrifugation and may employ selective purification processes that rely on the recognition of a unique envelope surface chemistry. Here we describe a novel method that fulfills these criteria. An immobilized metal affinity chromatography (IMAC) method was developed for the selective purification of vectors engineered to display a high-affinity binding peptide. Feasibility studies involving various transition metal ions (Cu2+, Zn2+, Ni2+, and Co2+) showed that cobalt had the most desirable features, which include a low level of interaction with either the normal virus envelope or contaminating DNA and proteins. The introduction of a cobalt-specific recognition element into the virus envelope may provide a suitable target for cobalt-dependent purification. To test this possibility, we engineered a peptide with affinity for immobilized cobalt in frame in the heparan sulfate binding domain of HSV-1 glycoprotein B, which is known to be exposed on the surface of the virion particle and recombined into the viral genome. By optimizing the IMAC loading conditions and reducing cobalt ion leakage, we recovered 78% of the tagged HSV-1 recombinant virus, with a >96% reduction in contaminating proteins and DNA.

Design of Affinity Tags for One‐Step Protein Purification from Immobilized Zinc Columns

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)‐iminodiacetic acid (Zn(II)‐IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)‐IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)‐IDA elution profile may be free of contaminant proteins, which can be exploited for one‐step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)‐IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.

Evaluation of Infection Parameters in the Production of Replication-Defective HSV-1 Viral Vectors

Herpes simplex virus type‐1 (HSV‐1) is a neurotrophic human pathogen that establishes life‐long latency in the nervous system. Our laboratory has extensively engineered this virus to retain the ability to persist in neurons without expression of lytic genes or disease phenotype. Highly defective, replication‐incompetent HSV mutants are thus potentially ideal for transfer of therapeutic transgenes to human nerves where long‐term therapy of nervous system disease may be provided. A prerequisite for using recombinant HSV vectors for therapeutic gene delivery to humans is the development of methods for large‐scale manufacture of HSV vectors. Here we report studies to identify infection parameters that result in high‐yield production of immediate early gene deletion mutant HSV vectors in complementing cells that supply the deleted essential viral functions in trans. Virus yield was correlated with various culture media conditions that included pH, glucose metabolism, and serum levels. The results demonstrated that systematic media exchange to remove lactate derived from high‐level glucose consumption, maintenance of tissue culture pH at 6.8, and the use of 5% fetal bovine serum gave the highest yield of infectious virus. The data indicate that these are important parameters to consider for high‐yield, large‐scale virus production.