Host Protein Contamination Research Articles

Comparison of Protein Gut Samples from Rhipicephalus spp. Using a Crude and an Innovative Preparation Method for Proteome Analysis

Tick populations are controlled through the application of chemical pesticides. However, the rise in chemical resistance has prompted the investigation of other control methods such as the use of tick vaccines. Proteomic analysis provides valuable information about the possible function and localization of proteins, as candidate vaccine proteins are often either secreted or localized on the cell-surface membrane. Progress in the utilization of proteomics for the identification of novel treatment targets has been significant. However, their use in tick-specific investigations is still quite novel, with the continual development of tick-specific methodologies essential. In this study, an innovative sample preparation method was utilized to isolate epithelial cells from tick midguts to identify the membrane-bound proteins. Proteomic analysis was conducted comparing crude and innovative sample preparation methods with 692 and 1242 tick-specific proteins, 108 and 314 surface proteins respectively, isolated from the midguts of semi-engorged Rhipicephalus microplus adult female ticks. This research reports a novel preparation protocol for the analysis of tick midgut proteins which reduces host protein contamination.

Improved protein-A chromatography for monoclonal antibody purification

Therapeutic monoclonal antibodies become the major product class within the biopharmaceutical market. Protein A as the first capture step is still dominant in current platforms for purification of monoclonal antibodies. In this study, we developed a new antibody harvest process that incorporates acidic treatment of cell harvest, demonstrating high process yield, improved clearance of host cell associated contaminants, like non-histone host cell protein, histone, DNA and heteroaggregates. Host protein contamination was reduced about 10-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Turbidity increase of eluted IgG upon pH neutralization was nearly eliminated. Residual levels of impurities in the protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. The mechanism of host cell associated contaminants removal during acidic treatment was also explored. After a polishing step by Capto adhere, host cell protein was reduced to less than 5 ppm, DNA less than 1 ppb, histone to undetectable level, heteroaggregates less than 0.01% with total IgG recovery around 87%. This efficient process can be easily integrated into current IgG purification platforms, and may overcome downstream processing challenges.

Advance chromatin extraction improves capture performance of protein A affinity chromatography

Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50–100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250–500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.

Non-immunospecific association of immunoglobulin G with chromatin during elution from protein A inflates host contamination, aggregate content, and antibody loss

Monoclonal IgG at pH 3.5 expressed a tendency to self-associate and associate non-specifically with surfaces, including the surfaces of precipitated chromatin heteroaggregates. The tendency was elevated with protein A-eluted IgG still in elution buffer (100mM acetate, pH 3.5). Association of IgG with chromatin elements under protein A elution conditions amplified host protein contamination of the elution fraction about 15-fold, caused formation of aggregates that persisted after pH neutralization, and imposed an approximate 5% loss on IgG recovery. Neutralization released eluted IgG from its low pH associations with chromatin and caused heteroaggregate remnants to associate into large particles easily removed by microfiltration. Most effective host contaminant clearance was achieved by filtration after neutralization to pH 5.5. All chromatin-mediated liabilities were suspended by extraction of chromatin heteroaggregates in advance of protein A.

Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance

Chromatin released from dead host cells during in vitro production of IgG monoclonal antibodies exists mostly in complex hetero-aggregates consisting of nucleosomal arrays (DNA+histone proteins), non-histone proteins, and aberrant forms of IgG. They bind immobilized protein A more aggressively than IgG, through their nucleosomal histone components, and hinder access of IgG to Fc-specific binding sites, thereby reducing dynamic binding capacity. The majority of host cell contaminants in eluted IgG are leachates from chromatin hetero-aggregates that remain bound to protein A. Formation of turbidity in eluted IgG during pH titration is caused by neutral-pH insolubility of chromatin hetero-aggregates. NaOH is required at 500mM to remove accumulated chromatin. A chromatin-directed clarification method removed 99% of histones, 90% of non-histone proteins, achieved a 6 log reduction of DNA, 4 log reduction of lipid-enveloped virus, and 5 log reduction of non-enveloped retrovirus, while conserving 98% of the native IgG. This suspended most of performance compromises imposed on protein A. IgG binding capacity increased ∼20%. Host protein contamination was reduced about 100-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Aggregates were reduced to less than 0.05%. Turbidity of eluted IgG upon pH neutralization was nearly eliminated. Column cleaning was facilitated by minimizing the accumulation of chromatin.

Antigen Specificity of the Humoral Immune Response to Mycoplasma haemofelis Infection

The aim of the present study was to characterize the antigenic specificity of the humoral immune response made by cats infected with the feline hemoplasma, Mycoplasma haemofelis. A crude M. haemofelis antigen preparation was prepared from red blood cells (RBCs) collected from a cat at the time of a high level of bacteremia. Plasma samples were collected from six cats before and after experimental infection with M. haemofelis, with regular sampling being performed from 15 to 149 or 153 days postinfection (dpi). Preinfection RBC membrane ghosts were prepared from these six cats and used to identify erythrocyte proteins that may have contaminated the M. haemofelis antigen preparation. The M. haemofelis antigen preparation comprised 11 protein bands. The immunodominant bands on Western blotting with infected cat plasma had molecular masses of 78, 68, 60, 48, and 38 kDa. Most cats (n = 5) had plasma antibody that reacted with at least one band (always including the one of 68 kDa) at 15 dpi, and all cats were seroreactive by 29 dpi. The maximum number of antibodies from an individual animal specific for an antigen was identified in plasma collected from 57 to 99 dpi. Contamination of the M. haemofelis antigen preparation with RBC membrane proteins was observed. The contaminating RBC proteins had molecular masses of from 71 to 72 kDa (consistent with band 4.2) and 261 and 238 kDa (consistent with spectrin), and these were recognized by all plasma samples. A range of M. haemofelis antigens is recognized by cats infected experimentally with the organism. These represent possible targets for immunoassays, but care must be taken to prevent false-positive results due to host protein contamination.

Extraction of Recombinant Dog Gastric Lipase from Transgenic Corn Seed

Several approaches were examined for extracting the relatively hydrophobic protein recombinant dog gastric lipase (rDGL) expressed in the endosperm of transgenic corn seed. The first approach used minimal processing of the seed before extraction (i.e. simple grinding of whole seed) followed by selective extraction to eliminate 72% of contaminant proteins without compromising rDGL recovery from the meal of whole grain. The second approach added defatting of the whole grain meal to reduce the amount of detergent in the subsequent step for extracting rDGL. The third approach incorporated dry-milling of the corn to recover an endosperm rich fraction, followed by extraction of this fraction. The dry milling strategy was most effective, resulting in recovery of 35 U rDGL/g of corn seed (50 U/g of endosperm) with a specific activity of 9 U/mg compared to 22 U and 3 U/mg for the first strategy and 36 U and 3.7 U/mg for the second. The reductions in host protein contamination and lower detergent levels of the endosperm route should simplify downstream purification steps.

Transduction of TAT-NF-YA Fusion Protein into K562 Human Chronic Myelogenous Leukemia Cells Upregulates HOXB4 Activity.

Protein transduction to redirect hematopoietic stem cell (HSC) fate for clinical therapy is an attractive alternative to genetic modification, in that the stem cell genome is not permanently modified. Indeed, published studies suggest that protein transduction with HOXB4, similar to retroviral overexpression of HoxB4, can increase HSC competitive repopulating ability. We have recently found that the trimeric transcription factor NF-Y, by modulation of its regulated subunit NF-YA, controls the expression of several HSC activating genes, including the Hox4 paralogs HoxB4, HoxC4, HoxD4 as well as p27, telomerase, Notch1, and LEF1. We have therefore now asked whether we can deliver NF-YA to hematopoietic cells by fusion with the protein transduction domain of the HIV-1 TAT protein, and modeled the molecular and biological activity of this protein by its effects on the HoxB4 promoter in vitro and in vivo. First, we designed and tested three fusion constructs expressing human NF-YA: with an N-terminal 6-histidine leader, a C-terminal 6xHis tag, and a GST tag at the N-terminus of NF-YA. Recombinant proteins bearing either the N- or C-terminal His-tag were poorly expressed under diverse growing and induction conditions, and were highly contaminated with bacterial host proteins. GST-fusion protein showed a much higher expression level and no host protein contamination, so all experiments were performed using GST-constructs. Next, electrophoretic mobility shift assays (EMSA) demonstrated that purified GST-TAT-NF-YA binds to HoxB4 promoter probes identically to endogenously translated NF-YA. NIH 3T3 fibroblasts, or K562 cells cultured in 0.1–2mM GST-TAT-NF-YA for 1.5h, but not without recombinant protein, showed bright cytoplasmic and nuclear fluorescence upon staining with anti-GST. To test the ability of GST-TAT-NF-YA to activate the HoxB4 promoter in live cells, GST-TAT-NF-YA's effect was measured both on transfected HOXB4 promoter reporter constructs and on endogenous HoxB4 mRNA. First, when GST-TAT-NF-YA was incubated with K562 cells electroporated with HoxB4 promoter/luciferase, along with plasmids encoding the NF-Ya partners NF-YB, NF-YC, USF1, USF2 (and Renilla luciferase for transfection control), normalized luciferase activity calculated as a % relative to control HoxB4-luc activity increased in an NF-YA concentration-dependent fashion. More pronounced effects were seen when GST-TAT-NF-YA protein was added to stably transfected HOXB4-luc K562 cell lines, with up to 2.7 fold increased HoxB4-luc activity versus control cells. Importantly, addition of GST-TAT-NF-YA protein to unmanipulated K562 cells increased expression of endogenous HoxB4 mRNA 2-2.5X, as measured by real-time PCR. Taken together, these data demonstrate that NF-YA protein can be biochemically, efficiently and functionally delivered to hematopoietic cells via TAT-mediated technology, providing a promising alternative to gene transfer to stimulate multiple HSC-activating genes.

Production of monoclonal antibodies to Tomato leaf curl Bangalore virus

SummaryPurified Tomato leaf curl Bangalore virus (ToLCBV) was injected into mice and the splenocytes were used for establishing hybridoma lines. Initial screening of culture supernatants showed that 13 lines produced antibody, and after further screening four produced functional monoclonal antibodies. Upon characterisation, these were found to be of low affinity, probably due to host protein contamination and poor yield of native virus in the original preparations. In order to circumvent these problems, the coat protein of ToLCBV was over‐expressed in Escherichia coli. Fusion experiments using recombinant coat protein as antigen yielded two primary hybridoma clones G11 and E4 that exhibited good affinity of binding to the antigen. Sub‐cloning yielded four monoclonal antibodies G11E7E7, G11E7G12, E4E2 and E4G6. G11E7E7 and G11E7G12 successfully detected ToLCBV in infected leaf extracts of tomato and Nicotiana benthamiana, viruliferous whiteflies and weed samples. These monoclonal antibodies could also detect other type III geminiviruses such as Pumpkin yellow vein mosaic virus and Bhendi yellow vein mosaic virus. Thus these monoclonal antibodies can be used for testing field‐collected samples.

Demonstration of a specific echinococcus multilocularis antigen in the supernatant of in vitro maintained protoscolices

Serodiagnosis of echinococcosis is still often met with difficulties resulting from unspecific reactions due to crude antigens which contain numerous host-derived proteins. In order to eliminate host protein contamination we produced Echinococcus multilocularis antigen by methods of in vitro technique: Evaginated protoscolices of Echinococcus multilocularis isolated from experimentally infected Mongolian gerbils ( Meriones unguiculatus) were maintained in RPMI 1640 medium. Although no serum proteins were added, protoscolices could be kept alive for more than 2 weeks. The supernatants harvested from protoscolices cultures were tested for their immunoreactivity against sera of patients with confirmed alveolar or cystic echinococcosis, cysticercosis, schistosomiasis or fascioliasis by means of SDS-PAGE and immunoblotting. A polypeptide band at about 62,000 mol. mass was identified which proved to be specifically immunoreactive with sera from patients with alveolar echinococcosis, whereas sera from patients with cystic echinococcosis or with other helminthic infections as well as sera from healthy blood-donors did not react with this polypeptide band. Thus, the use of supernatant antigen in immunoblotting technique allows a serological differentiation between infections with Echinococcus multilocularis and those with Echinococcus granulosus and provides an accurate diagnosis of alveolar echinococcosis.

Analysis of structural polypeptides of purified human cytomegalovirus

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.