Protein Chromatography Research Articles

Effect of flow behavior in extra-column volumes on the retention pattern of proteins in a small column

Experimental and theoretical analysis of deformation of band profiles in extra-column volumes (ECV) was performed, and its influence on the retention pattern of proteins in a small chromatographic column was quantified. Several macromolecule and small-molecule compounds, and their mixtures were eluted from a chromatographic system in the absence and presence of the column. The peak deformation in ECV was attributed to non-uniform velocity distribution in the radial direction in connecting capillaries. The phenomenon enhanced with increasing molecular weight of the model compound, when radial diffusion dominated the mechanism of band spreading. The band shape was also affected by the geometry of the injection system used, i.e., an injection loop capillary or a superloop. The phenomenon vanished for a small molecule compound, for which plug flow conditions could be established. The difference in flow behaviour of the macromolecule and small-molecule compounds caused them to migrate with different velocities in ECV, which resulted in partial separation of their bands. The ECV effect influenced the retention behaviour of macromolecules in a small column; it caused tailing of peaks and asymmetry of breakthrough curves. To describe the elution profiles in ECV and in the column, a mathematical model was used which accounted for non-ideality of the flow pattern. The model reproduced accurately band profiles of macromolecules within a range of relatively low velocities, typical however for protein chromatography.

Poly(N,N-dimethylaminopropyl acrylamide)-grafted Sepharose FF: A new anion exchanger of very high capacity and uptake rate for protein chromatography

Ion exchangers of high binding capacity and uptake rate are desired for high performance purification of proteins by ion exchange chromatography. For this purpose, we have herein developed a new anion exchanger by grafting N,N-Dimethylaminopropyl acrylamide (DMAPAA) onto Sepharose FF by atom transfer radical polymerization. Six DMAPAA-grafted Sepharose FF resins of the same grafting density but different chain lengths (ionic capacities, ICs) were prepared, and named as FF-pDMAPAAn (n denotes IC value, 65–831 mmol/L). Bovine serum albumin (BSA) adsorption and its chromatography were studied to evaluate the anion exchange resins. It was found that the equilibrium protein adsorption capacity (qm) increased rapidly from 91 mg/mL at IC = 65 mmol/L till reaching a peak value (368 mg/mL) at IC = 458 mmol/L and then decreased slightly with further increasing IC (chain length) to 831 mmol/L. The uptake rate, De/D0, defined as the ratio of effective diffusion coefficient (De) to free diffusivity of the protein (D0), also presented similar changes as a function of IC. Namely, De/D0 increased from 0.25 to 2.45 in the IC range of 65 to 458 mmol/L, kept almost unchanged from 458 to 573 mmol/L and then decreased slightly with further increase of IC to 831 mmol/L. Therefore, FF-pDMAPAA458 presented the best performance of the six resins for BSA adsorption, possessing very high qm and De/D0 values (368 mg/mL and 2.45, respectively). The resin was thus extensively characterized in the effect of ionic strength and column chromatography. The qm and De/D0 of FF-pDMAPAA458 were much higher than other anion exchangers reported in literatures and kept over 230 mg/mL and 2.3, respectively, at salt concentrations up to 100 mmol/L. Therefore, the dynamic binding capacity of the FF-pDMAPAA458 column was remarkably higher than Q Sepharose FF and other two typical polymer-grafted anion exchangers reported in literatures in the salt concentration range, and kept as high as 180–220 mg/mL in the superficial flow velocity rang of 150–1350 cm/h. Taken together, the results demonstrated that FF-pDMAPAA resins of appropriate ionic capacities are promising for high-performance protein chromatography.

Protein adsorption to poly(allylamine)-modified Sepharose FF: Influences of polymer size and partial charge neutralization

Recently, poly(allylamine) (PAA) of 17.5 and 900 kDa were grafted onto Sepharose FF gel to study protein adsorption and chromatography. In order to further investigate the influences of polymer size and partial charge neutralization, PAAs of 160 and 450 kDa were chosen to synthesize two more types of ion-exchangers (denoted as FF-PAA-M and FF-PAA-ML, respectively). Protein adsorption capacity (qm) of these resins showed a monotonic increasing trend till the maximum ionic capacity (IC) available (970–990 mmol/L), while the uptake rates (De/D0) showed a decreasing-then-increasing trend. The maximum qm and De/D0 reached 328 mg/mL and 0.715, respectively, with FF-PAA-M990 (IC = 990 mmol/L). Partial charge neutralization by modification of FF-PAA-M990 with acetate produced four charge-reduced resins (FF-PAA-Rs), of which the lowest IC reached 210 mmol/L. Both the qm and De/D0 of FF-PAA-Rs presented an increasing-then-decreasing trend with reducing the charge density. The maximum qm obtained with FF-PAA-R860 (IC = 860 mmol/L) was 40% higher than FF-PAA-M990, and all the charge-reduced resins presented 2–3 times higher uptake rates than FF-PAA-M990. Both the qm and De/D0 of FF-PAA-Rs decreased sharply with increasing salt concentration, indicating their increased salt-sensitivities. The FF-PAA-R860 and FF-PAA-R710 columns presented much higher dynamic binding capacities than the FF-PAA-M990 column. FF-PAA-M990 and the charge-reduced resins were suitable for protein chromatography handling feedstocks containing different salt concentrations due to their different sensitivities to ionic strength.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

BackgroundAnimal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads’ secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules.MethodsTotal RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman’s degradation.ResultsWe were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion.ConclusionsWe identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.

Adsorption of IgG and BSA on Two Chromatographic Resins—Poly(ethylenimine)-4FF Resin and Tetrapeptide-poly(ethylenimine)-4FF Resin

Poly(ethylenimine) (PEI) as a long flexible chain has been well used in protein chromatography with the advantages of high spatial utilization and “chain delivery” effect. In this work, two resins, i.e., poly(ethylenimine)-4FF resin (PEI-4FF resin) and Ac-YFRH-PEI-4FF resin by grafting tetrapeptide to PEI-4FF resin, were prepared for the protein adsorption and chromatographic separation. The pH-dependent adsorption properties of PEI-4FF resin and Ac-YFRH-PEI-4FF resin were discovered by getting the data of adsorption capacities of bovine serum albumin (BSA) and immunoglobulin G (IgG) under different pH conditions and buffer systems. The results showed high binding capacities of BSA on PEI-4FF resins across a range of pH values (5.0–8.9) with almost very little adsorption of IgG over a pH range from 5.0 to 8.0, indicating that IgG could be separated by flow-through method using PEI-4FF resin. Since PEI has the advantage of having many flexible branched chains and Ac-YFRH (Ac-Try-Phe-Arg-His) as a peptide l...

Protein adsorption to poly(ethylenimine)-modified sepharose FF: VII. Complicated effects of pH

Previously, we have studied protein adsorption behaviors on a series of poly(ethylenimine) (PEI)-grafted Sepharose FF, and a critical ionic capacity of PEI-grafted resins was observed, above which both protein adsorption capacity (q) and effective pore diffusivity (De) increased drastically. Moreover, reducing the charge density of the PEI-grafted resins from an ionic capacity of 740 mmol/L (FF-PEI-L740) to 440 mmol/L (FF-PEI-R440) by neutralization of the amino groups of PEI chains with sodium acetate brought out a three-fold increase of De value at pH 8. In this work, FF-PEI-L740 and FF-PEI-R440 were selected to investigate the complicated effects of pH on protein adsorption behavior using bovine serum albumin (BSA) as the model protein. It was found that, for FF-PEI-L740, both the q and De values decreased significantly when pH decreased from 9 to 5.5, and then increased dramatically at pH 5, and finally decreased remarkably at pH 4.5. The results were considered due to the following causes: The decrease of pH from 9 to 5.5 led to a greatly increased electrostatic repulsion between the PEI chains due to the increased dissociation degree of PEI, which caused the increase in steric hindrance effects and decrease in the bound protein transport by chain delivery. At pH 5, which was near the pI of BSA (∼4.9), the presence of few protein charges greatly decreased the electrostatic hindrance effect on protein transport and the high charge density of FF-PEI-L740 provided extensively accessible binding sites and facilitated the happening of chain delivery as well. At pH 4.5, the net charge of BSA shifted to positive, likely charged with PEI, so the overall electrostatic repulsion greatly hindered protein uptake. For FF-PEI-R440, whose charge density was much lower than FF-PEI-L740, its q and De values even increased mildly when pH decreased from 8 to 5.5, because its slightly-increased charge density did not cause much increased electrostatic and steric hindrance effects but provided more binding sites for protein adsorption. At pH 5, the few charges of BSA and the low charge density of FF-PEI-R440 could not afford enough electrostatic interaction for protein binding, resulting in significantly decreased q and De values. Besides, column breakthrough experiments revealed that FF-PEI-R440 kept high dynamic binding capacity (DBC) (>120 mg/mL) at pH 5.5–8, while FF-PEI-L740 offered high DBC (>80 mg/mL) at pH 5, 7 and 8 with a higher salt concentration (100 mmol/L NaCl). These findings demonstrated the excellence of FF-PEI-L740 and FF-PEI-R440 in different conditions, and would help in the design and selection of suitable resins for high-performance protein chromatography.

Grafting glycidyl methacrylate-iminodiacetic acid conjugate to Sepharose FF for fabrication of high-capacity protein cation exchangers

This work was designed to acquire novel high-capacity cation exchangers by grafting glycidyl methacrylate (GMA)-iminodiacetic acid (IDA) conjugate (GMA-IDA) to Sepharose FF. Six GMA-IDA-grafted resins of the same grafting density but different chain lengths (ionic capacities, ICs) were synthesized and denoted as FF-Br2-pG-In (n denotes for IC value). Lysozyme adsorption and chromatography were studied to evaluate the cation exchangers. It was found that resins of moderate IC values (FF-Br2-pG-I480 and FF-Br2-pG-I590) afforded lysozyme adsorption capacities higher than 300 mg/mL at 50 and 100 mmol/L NaCl, over three times higher than the commercial resin CM Sepharose FF. Beside, the uptake rate (De/D0) of lysozyme decreased with increasing IC at 50 mmol/L NaCl because of the increased polymer chain length that limited surface diffusion of the bound protein. Protein adsorption capacities of FF-Br2-pG-I480 and FF-Br2-pG-I590 kept higher than that of CM Sepharose FF in the salt concentration range of 0–150 mmol/L, and the uptake rates on the two resins increased with increasing salt concentration. Therefore, the dynamic binding capacities of them were remarkably higher than that of CM Sepharose FF and kept over 70 mg/mL at the salt concentration up to 150 mmol/L. The results demonstrated that GMA-IDA-grafted resins are promising for use as high-capacity protein chromatography.

Fabrication of high-capacity cation-exchangers for protein adsorption: Comparison of grafting-from and grafting-to approaches

In this work, we have synthesized two polymer-grafted cation exchangers: one via the grafting-from approach, in which sulfopropyl methacrylate (SPM) is grafted through atom transfer radical polymerization onto Sepharose FF (the thus resulting exchanger is referred as Sep-g-SPM), and another via the grafting-to approach, in which the polymer of SPM is directly coupled onto Sepharose FF (the thus resulting exchanger is called as Sep-pSPM). Protein adsorption on these two cation exchangers have been also investigated. At the same ligand density, Sep-g-SPM has a larger accessible pore radius and a smaller depth of polymer layer than SeppSPM, due to the controllable introduction of polymer chains with the regular distribution of the ligand. Therefore, high-capacity adsorption of lysozyme and γ-globulin could be achieved simultaneously in Sep-g-SPM with an ionic capacity (IC) of 308 mmol$L–1. However, Sep-pSPM has an irregular chain distribution and different architecture of polymer layer, which lead to more serious repulsive interaction to proteins, and thus Sep-pSPM has a lower adsorption capacity for γ-globulin than Sep-g-SPM with the similar IC. Moreover, the results from protein uptake experiments indicate that the facilitated transport of adsorbed γ-globulin occurs only in Sep-pSPM and depends on the architecture of polymer layers. Our research provides a clear clue for the development of high-performance protein chromatography.

Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry.

Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and β‐lactoglobulin to Toyopearl Butyl‐650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases. Experiments were carried out at two different salt concentrations and five different temperatures. Protein unfolding, as indicated by large changes of molar enthalpy of adsorption Δh ads, was observed to be dependent on temperature and salt concentration. Δh ads were significantly higher for bovine serum albumin and ranged from 578 (288 K) to 811 (308 K) kJ/mol for 1.2 mol/kg ammonium sulfate. Δh ads for β‐lactoglobulin ranged from 129 kJ/mol (288 K) to 186 kJ/mol (308 K). For both proteins, Δh ads increased with increasing temperature. The influence of salt concentration on Δh ads was also more pronounced for bovine serum albumin than for β‐lactoglobulin. The comparison of retention analysis evaluated by the van't Hoff algorithm shows that beyond adsorption other processes occur simultaneously. Further interpretation such as unfolding upon adsorption needs other in situ techniques.

Prediction tool for loading, isocratic elution, gradient elution and scaling up of ion exchange chromatography of proteins

An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media.

Online Hydrophobic Interaction Chromatography-Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies.

Therapeutic monoclonal antibodies (mAbs) are an important class of drugs for a wide spectrum of human diseases. Liquid chromatography (LC) coupled to mass spectrometry (MS) is one of the techniques in the forefront for comprehensive characterization of analytical attributes of mAbs. Among various protein chromatography modes, hydrophobic interaction chromatography (HIC) is a popular offline nondenaturing separation technique utilized to purify and analyze mAbs, typically with the use of non-MS-compatible mobile phases. Herein we demonstrate for the first time, the application of direct HIC-MS and HIC-tandem MS (MS/MS) with electron capture dissociation (ECD) for analyzing intact mAbs on quadrupole-time-of-flight (Q-TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, respectively. Our method allows for rapid determination of relative hydrophobicity, intact masses, and glycosylation profiles of mAbs as well as sequence and structural characterization of the complementarity-determining regions in an online configuration.

Calixarene-immobilized monolithic cryogels for preparative protein chromatography

New cation exchanger monolithic stationary phases were prepared by immobilization of three different calixarene derivatives (i.e. tetracarboxylate calix[4]arene, CLX-COO, tetrasulfonate calix[4]arene, CLX-SO3, and tetraphosphonate calix[4]arene, CLX-PO4) onto a monolithic cryogel support (i.e. poly(2-hydroksyethylmethacrilate-co-glycidyl methacrylate, P) and investigated with respect to preparative protein chromatography. The obtained monoliths were characterized through various techniques such as FTIR spectroscopy, isoelectric point measurements, titrimetric analyses, and mercury intrusion porosimetry. Protein retention was investigated using some model proteins (i.e. lysozyme, cytochrome c, and ɑ-chymotrypsinogen A, human serum albumin, and myoglobin), and the role of modifier (i.e. NaCl) concentration and pH was thoroughly analyzed under isocratic and gradient elution conditions. Overloading experiments were also conducted to study dynamic adsorption capacity and the obtained values were found to be ranging between 3 and 8 mg/mL depending on the type of calixarene molecule. Hence, higher or comparable protein adsorption capacities were seen to be applicable on calixarene-immobilized cryogels when compared to any other functionalized cryogels in the literature. Combined with the favorable properties of these monoliths, with respect to mass transport of large molecules, these results qualify calixarene functionalized monolithic cryogels as promising stationary phases for protein preparative purification.

Starch Synthase IIa-Deficient Mutant Rice Line Produces Endosperm Starch With Lower Gelatinization Temperature Than Japonica Rice Cultivars.

The gelatinization temperature of endosperm starch in most japonica rice cultivars is significantly lower than that in most indica rice cultivars. This is because three single nucleotide polymorphisms in the Starch synthase (SS) IIa gene in japonica rice cultivars (SSIIaJ) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ≤ 12 compared to indica rice cultivars (SSIIaI). SSIIa forms a trimeric complex with SSI and starch branching enzyme (BE) IIb in maize and japonica rice, which is likely important for the biosynthesis of short and intermediate amylopectin chains (DP ≤ 24) within the amylopectin cluster. It was unknown whether the complete absence of SSIIa further increases amylopectin short chains and reduces gelatinization temperature and/or forms altered protein complexes due to the lack of a suitable mutant. Here, we identify the SSIIa-deficient mutant rice line EM204 (ss2a) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to N-methyl-N-nitrosourea mutagenesis. The SSIIa gene in EM204 was mutated at the boundary between intron 5 and exon 6, which generated a guanine to adenine mutation and resulted in deletion of exon 6 in the mRNA transcript. SSIIa activity and SSIIa protein in developing endosperm of EM204 were not detected by native-PAGE/SS activity staining and native-PAGE/immunoblotting, respectively. SSIIa protein was completely absent in mature seeds. Gel filtration chromatography of soluble protein extracted from developing seeds showed that the SSI elution pattern in EM204 was altered and more SSI was eluted around 300 kDa, which corresponds with the molecular weight of trimeric complexes in wild type. The apparent amylose content of EM204 rice grains was higher than that in its parent Kinmaze. EM204 also had higher content of amylopectin short chains (DP ≤ 12) than Kinmaze, which reduced the gelatinization temperature of EM204 starch by 5.6°C compared to Kinmaze. These results indicate that EM204 starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.

Sequential alginate grafting and sulfonation significantly improve the performance of alginate-grafted Sepharose FF for protein chromatography

Alginate (Alg)-grafted Sepharose FF with an ionic capacity (IC) of 230 mmol/L (Alg-FF-230) was first prepared and then sulfonated to synthesize two cation exchangers of higher ICs (270 and 370 mmol/L, denoted as S-Alg-FF-IC). It was found that lysozyme adsorption capacity increased with increasing IC value, but the uptake rate (De/D0) on S-Alg-FF-370 was small (0.070) at 0 mmol/L NaCl because of the strong binding that limited surface diffusion of the bound protein. However, the dynamic binding capacity (DBC) of S-Alg-FF-370 was much higher in the salt concentration range of 0–150 mmol/L than the lower IC resins due to its high adsorption capacity and increased uptake rate. Especially at 150 mmol/L NaCl, the DBC of S-Alg-FF-370 was about 7.3 times higher than Alg-FF-230 and kept over 70 mg/mL at the flow rate up to 1350 cm/h. The results proved the high salt-tolerant feature of S-Alg-FF-370. By contrast, S-Alg-FF-270 showed higher DBC values than S-Alg-FF-370 in the same flow rate range at 50 mmol/L NaCl. Hence, S-Alg-FF-270 and S-Alg-FF-370 displayed different properties that would make them favorable for use in protein purification at different salt concentrations. The work thus proved that the sequential alginate grafting and sulfonation strategy is promising for creation of ion exchangers for high-performance and cost-effective purification of proteins from feedstocks of different ionic strengths.

Analyses of starch biosynthetic protein complexes and starch properties from developing mutant rice seeds with minimal starch synthase activities

BackgroundStarch is the major component of cereal grains and is composed of essentially linear amylose and highly branched amylopectin. The properties and composition of starch determine the use and value of grains and their products. Starch synthase (SS) I, SSIIa, and SSIIIa play central roles in amylopectin biosynthesis. These three SS isozymes also affect seed development, as complete loss of both SSI and SSIIIa under reduced SSIIa activity in rice lead to sterility, whereas presence of minimal SSI or SSIIIa activity is sufficient for generating fertile seeds. SSs, branching enzymes, and/or debranching enzymes form protein complexes in cereal. However, the relationship between starch properties and the formation of protein complexes remain largely unknown. To better understand this phenomenon, properties of starch and protein complex formation were analyzed using developing mutant rice seeds (ss1L/ss2aL/ss3a) in which all three major SS activities were reduced.ResultsThe SS activity of ss1L/ss2aL/ss3a was 25%–30% that of the wild-type. However, the grain weight of ss1L/ss2aL/ss3a was 89% of the wild-type, 55% of which was starch, showing considerable starch synthesis. The reduction of soluble SS activity in ss1L/ss2aL/ss3a resulted in increased levels of ADP-glucose pyrophosphorylase and granule-bound starch synthase I, which are responsible for substrate synthesis and amylose synthesis, respectively. Together, these features led to an increase in apparent amylose content (34%) in ss1L/ss2aL/ss3a compared with wild-type (20%). Gel filtration chromatography of the soluble proteins in ss1L/ss2aL/ss3a showed that the majority of the starch biosynthetic enzymes maintained the similar elution patterns as wild-type, except that the amounts of high-molecular-weight SSI (> 300 kDa) were reduced and SSIIa of approximately 200–300 kDa were present instead of those > 440 kDa, which predominate in wild-type. Immuno-precipitation analyses suggested that the interaction between the starch biosynthetic enzymes maybe reduced or weaker than in wild-type.ConclusionsAlthough major SS isozymes were simultaneously reduced in ss1L/ss2aL/ss3a rice, active protein complexes were formed with a slightly altered pattern, suggesting that the assembly of protein complexes may be complemented among the SS isozymes. In addition, ss1L/ss2aL/ss3a maintained the ability to synthesize starch and accumulated less amylopectin and more amylose in starch.

Promoting hypothesis‐driven thinking in the undergraduate biochemistry lab

We have developed a Course‐based Undergraduate Research Experience (CURE), which is built on a combination of traditional wet lab techniques (protein expression, chromatography, electrophoresis, protein and activity assays) and bioinformatics tools (sequence and structure alignment, visualization and molecular docking). Two major factors have led to this project ‐ the initiative of students and the influence of collaborators. Students in our research groups at the Rochester Institute of Technology and Dowling College developed the ProMOL plugin for PyMOL, which enabled us to perform active site alignments between two enzymes. They then enhanced ProMOL to include a library of one thousand enzyme active site templates, which they used to predict functions for unannotated protein structures in the Protein Data Bank. They added sequence and backbone alignments to our bioinformatics approach, then insisted on moving into the wet lab to validate the functions they had predicted. In this process, we saw our students make such remarkable strides as scientists that we decided to replicate this approach in an undergraduate biochemistry teaching lab. The project, which we call BASIL, for Biochemistry Authentic Scientific Inquiry Lab, now involves thirteen faculty members teaching the lab on eight campuses. We are now engaged in a longer term assessment and evaluation of student growth as scientists in this teaching lab setting.Support or Funding InformationThis work is supported by the National Science Foundation under Awards #1503811 and #1709170, the National Institute of General Medical Sciences under Award #78077, and the campuses represented in the BASIL project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acetonitrile as solvent for protein interaction analysis

Dimethyl sulfoxide (DMSO) is routinely used to dissolve hydrophobic and aromatic substances in drug screening. The dissolved substances are then diluted, e.g., 100-fold, into aqueous media containing their target molecules. Such a practice may be suitable for biological evaluation of the drug substances. However, structural evaluation using spectroscopic techniques, in particular far UV measurements, may be hampered by strong absorbance of DMSO. Here, I have tested acetonitrile (ACN) that is almost exclusively used for reverse phase chromatography of proteins and small organic molecules. Using a small aromatic/hydrophobic molecule, dodecyl-gallate, and bovine serum albumin as a model system, a side-by-side comparison was made for DMSO and ACN with regard to dissolution capability and far UV circular dichroism (CD) performance.

High uptake rate and extremely salt-tolerant behavior of protein adsorption to 900 kDa poly(allylamine)-modified Sepharose FF

This work reports poly(allylamine) of 900 kDa (PAA-L) as a polymeric ligand for protein ion-exchange chromatography. PAA-L-modified Sepharose FF with 9 ionic capacities (ICs) from 80 to 880 mmol/L were prepared and bovine serum albumin adsorption and chromatography were investigated. With increasing IC, protein adsorption capacity increased somewhat in the IC range of 80–420 mmol/L, and then increased twice in the IC range of 420–740 mmol/L, and finally kept unchanged till 880 mmol/L; the uptake rate (De/D0) increased about 14 times (from 0.029 to 0.42) in the IC range of 420–700 mmol/L, and finally kept almost unchanged till 880 mmol/L. The maximum adsorption capacity and uptake rate of the resins reached 336 mg/mL and 0.42, which were 24% and 2.5 times respectively larger than those of 17.5 kDa PAA-grafted resins. The selected PAA-L resin of IC = 740 mmol/L was salt-tolerant up to 750 mmol/L NaCl. The dynamic binding capacity (DBC) of this resin showed a maximum of 142 mg/mL at 350 mmol/L NaCl, and kept over 90 mg/mL in the range of 150–500 mmol/L NaCl. Linear gradient elution on the column of this resin proved high protein recovery (>97%) and stability of the column. Taken together, the results indicate that the PAA-L resin was a promising anion exchanger for high-performance protein chromatography for use at extended ionic strengths.

Reversible Concanavalin A (Con A) ligands immobilization on metal chelated macroporous cellulose monolith and its selective adsorption for glycoproteins

The present work deals with the development of novel affinity monolith with reversible protein ligands for protein chromatography. As for the formation of reversible ligands, Concanavalin A (Con A) is chelated with Cu(II)-iminodiacetic acid (IDA) immobilized macroporous cellulose monolith (MCM) for glycoprotein adsorption. The reversible immobilization is realized by Cu ions, which bridge affinity ligands and support by strong chelation interaction. The fabrication process of reversible Con A immobilized adsorbent is studied, especially with regards to the effect of synthesis conditions on the ligands immobilization. The adsorption behavior is then evaluated to elucidate the potential of Con A-Cu(II)-IDA-MCM for protein chromatography. It reveals that the static adsorption capacity and dissociation constant of glucose oxidase (GOD) on Con A-Cu(II)-IDA-MCM are determined to be 17.4 ± 0.6 mg mL−1 and 0.055 ± 0.011 mg mL−1 by Langmuir model. With frontal analysis, the dynamic binding capacity of GOD at 10% breakthrough point is about 11.4 ± 1.0 mg mL−1 and changes less with an increase of flow velocity from 0.2 to 1.0 mL min−1. Moreover, Con A-Cu(II)-IDA-MCM displays weak nonspecific adsorption for the impurities and is able to successfully enrich glycoprotein ovalbumin (OVA) from diluted chicken egg white. In addition, Con A-Cu(II)-IDA-MCM exhibits excellent stability by the repeated adsorption/desorption operations. By taking these advantages of high adsorption capacity, excellent specificity and structure stability, the prepared affinity adsorbent of Con A-Cu(II)-IDA-MCM has great potential for high performance protein chromatography.

In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography

Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q2 values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q2 of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques.