Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

Process evaluations and economic analyses of recombinant human lysozyme and hen egg-white lysozyme purifications

Human lysozyme and hen egg-white lysozyme have antibacterial, antiviral, and antifungal properties with numerous potential commercial applications. Currently, hen egg-white lysozyme dominates low cost applications but the recent high-level expression of human lysozyme in rice could provide an economical source of lysozyme. This work compares human lysozyme and hen egg-white lysozyme adsorption to the cation exchange resin, SP-Sepharose FF, and the effect of rice extract components on lysozyme purification. With one exception, the dynamic binding capacities of human lysozyme were lower than those of hen egg-white at pH 4.5, 6, and 7.5 with ionic strengths ranging from 0 to 100 mM (5-20 mS). Ionic strength and pH had a similar effect on the adsorption capacities, but human lysozyme was more sensitive to these two factors than hen egg-white lysozyme. In the presence of rice extract, the dynamic binding capacities of human and hen egg-white lysozymes were reduced by 20-30% and by 32-39% at pH 6. Hen egg-white lysozyme was used as a benchmark to compare the effectiveness of human lysozyme purification from transgenic rice extract. Process simulation and cost analyses for human lysozyme purification from rice and hen egg-white lysozyme purification from egg-white resulted in similar unit production costs at 1 ton per year scale.     

Expression of natural antimicrobial human lysozyme in rice grains

In the present study, we explored the expression of human lysozyme in maturing rice grains. Particle bombardment-mediated transformation was utilized to deliver the codon-optimized structural gene for human lysozyme to the callus of rice cultivar Taipei 309. Lysozyme expression is controlled by the promoter and signal peptide sequence for rice storage protein Glutelin 1. A total of 33 fertile plants were regenerated from independent transformation events and 12 of them with significant expression levels of lysozyme were advanced to further generations. The transgenes were characterized by PCR and Southern blot analysis. Segregation analysis indicated a typical Mendelian 3: 1 inheritance, suggesting a single locus or closely linked loci of gene insertion. The expression levels of lysozyme reached 0.6% of the brown rice weight or 45% of soluble proteins. Seven transgenic breeding lines have been selected and followed over six generations. Lysozyme expression levels were maintained in all generations. Biochemical, biophysical and functional comparisons of native and recombinant human lysozyme revealed identical N-terminal sequence, molecular weight, pI and specific activity. Similar thermal and pH stability was observed for lysozyme from two sources. Furthermore, similar bactericidal activity was displayed towards a laboratory strain of E. coli. The possibility of improving medical and nutritional quality of infant formulas and baby foods with rice flour or rice extract containing recombinant human lysozyme is discussed.     

Expression of human lysozyme mRNA in the mammary gland of transgenic mice

Owing to its inherent antimicrobial effect and positive charge, the expression of human lysozyme in bovine milk could be beneficial by altering the overal microbial level and the functional and physical properties of the milk. We have used transgenic mice as model systems to evaluate the expression of human lysozyme containing fusion gene constructs in the mammary gland. Expression of human lysozyme was targeted to the mammary gland by using the 5 promoter elements of either the bovine (line B mice) or _s1 (line H mice) casein genes coupled to the cDNA for human lysozyme. Expression of human lysozyme mRNA was not found in mammary tissue from any of line B mice. Tissues were analysed from six lines of H mice and two, H6 and H5, were found to express human lysozyme mRNA in the mammary gland at 42% and 116%, respectively, of the levels of the endogenous mouse whey acidic protein gene. At peak lactation, female mice homozygous for the H5 and H6 transgene have approximately twice the amount of mRNA encoding human lysozyme as hemizygous animals. Expression levels of human lysozyme mRNA in the mammary gland at time points representing late pregnancy, early, peak and late lactation corresponded to the profile of casein gene expression. Human lysozyme mRNA expression was not observed in transgenic males, virgin females or in the kidney, liver, spleen or brain of lactating females. A very low level of expression of human lysozyme mRNA was observed in the salivary gland of line H5.     

Characterization of superporous cellulose matrix for high‐throughput adsorptive purification of lysozyme

Protein purification essentially requires macroporous adsorbents matrices, which can provide high efficiency in packed bed and expanded bed (EB) even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the superpores. Rigid spherical superporous adsorbent beads with high carboxyl group density were prepared by crosslinking of cellulose. The matrix (diameter: 100–300 μm, mean pore size: 1–3 μm, pore volume: 57–59%, and bulk density: ～1,438 kg/m³) could be used in packed bed as well as EB for purification of various biomolecules. Attempts were made to use indigenously developed rigid, superporous crosslinked cellulose adsorbent for high‐throughput purification of lysozyme from chicken egg white's extract. A typical adsorption isotherm for lysozyme in crude was well correlated with the Langmuir isotherm model. Two maxima of binding capacity on celbeads bearing carboxymethyl (celbeads‐CM) group for lysozyme were observed at pH 4.5 and 7.5. Uptake kinetics showed that the diffusivity of lysozyme was 100 times higher than conventional matrices. Such superporous matrix can be used for high‐throughput purification of proteins from crude feedstocks and is reflected in leveling off of height equivalent to theoretical plate vs. flow curve after threshold velocity. Optimization of binding and elution conditions resulted in overall purification of lysozyme in a high yield and purity of 98.22 and 98.8%, respectively, with purification factor of 51.54 in a single step. The overall productivity (14.21 kg/m³ h) and specific activity (2.2 × 10⁵ U/mg) were higher than that obtained with traditional particulate resins. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Purification of human lysozyme from milk and pancreatic juice

Human milk lysozyme was purified by heparin-Sepharose affinity chromatography and Sepharose 4B gel-permeation chromatography. This procedure was also found applicable to the purification of human pancreatic juice lysozyme. Double-diffusion analyses indicated that human milk lysozyme was immunochemically identical to human saliva and human pancreatic juice lysozyme. Based on the identity of the N-terminal 10-amino-acid-residue sequence analyzed, it was suggested that human milk lysozyme and human pancreatic juice lysozyme are identical molecular entities.     

低无机离子稻糠植酸提取方法的研究

采用醋酸解离并结合加热使植酸从种皮的复合盐中释放、石灰乳沉淀、乳酸酸化植酸钙、碱解再用离子交换脱无机离子,结合流程采用活性炭脱色的方法制备植酸.与同类方法相比,所得植酸纯度较高(68%～72%),总回收率达到70%以上;重复性及产品可溶性好,无非目的成分沉淀;色泽透明清亮,对优化粮油深加工的流程,提高产品质量有积极意义.     

Sulfanilic acid‐modified chitosan mini‐spheres and their application for lysozyme purification from egg white

A cation exchange matrix with zwitterionic and multimodal properties was synthesized by a simple reaction sequence coupling sulfanilic acid to a chitosan based support. The novel chromatographic matrix was physico‐chemically characterized by ss‐NMR and ζ potential, and its chromatographic performance was evaluated for lysozyme purification from diluted egg white. The maximum adsorption capacity, calculated according to Langmuir adsorption isotherm, was 50.07 ± 1.47 mg g^?1 while the dissociation constant was 0.074 ± 0.012 mg mL^?1. The process for lysozyme purification from egg white was optimized, with 81.9% yield and a purity degree of 86.5%, according to RP‐HPLC analysis. This work shows novel possible applications of chitosan based materials. The simple synthesis reactions combined with the simple mode of use of the chitosan matrix represents a novel method to purify proteins from raw starting materials. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:387–396, 2018     

Harvesting of Arthrospira platensis by flocculation with phytic acid from rice bran

ABSTRACT

The recovery of algal biomass is one of the critical steps involved in the commercial production of beneficial metabolites from Arthrospira platensis. Efficient and safe harvesting methods that do not sacrifice quality of final product are important for commercial application. Phytic acid (PA) is a natural non-toxic phytochemical widely distributed in plant tissues. Effect of PA from rice bran on the growth, trichome morphology such as spiral number and algal filament length, and harvesting efficiency of A. platensis were investigated. Cells aggregated into large cell flocs after the addition of PA in the medium, and algal spiral number and filament length increased. UV-vis spectra indicated the interactions between PA and algal cells. Adding PA at stationary growth phase is a good strategy for harvesting, since no adverse effect to biomass growth and harvesting efficiency. Harvesting efficiency of 95.69% at 0.5% (v/v) PA was superior to other conventional harvesting methodologies.     

Simple purification of aromatic -amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic -amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an M_r of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated -3,4-dihydroxyphenyl-alanine, -5-hydroxytryptophan, and -threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.     

Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures

We investigated the possibility of producing the therapeutic recombinant cytokine, Interferon-gamma (IFN-), in transgenic rice cell (Oryza sativa, cultivar TNG67) suspension cultures. We tested expression of two vector constructs, each harboring an Amy3 leader peptide and a C-terminus His 6 tag fused to a human IFN- cDNA, one driven by a sucrose-starvation inducible promoter (rice Amy3 promoter) and the other by a constitutive maize ubiquitin promoter, in rice cell suspensions, introduced via Agrobacterium tumefaciens. There was a significant difference in the amounts of recombinant IFN- protein produced by the Ups and Amy cell lines, as cytosolic and secretory proteins respectively. Immunological analysis of IFN- recombinant protein conferred a dose-dependent anti-dengue virus activity in human A549 cells, similar to the commercial product. We discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant IFN-.     

Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography

Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion‐exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10⁸ L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:864–871, 2014     

人源脂肪酸合酶在酿酒酵母中的表达纯化和结构的初步分析

[背景] 聚酮类化合物在医药领域有重要的应用,相关药物研发依赖聚酮合酶多变的结构认知,人源脂肪酸合酶的组成结构和催化机制与聚酮合酶相近,研究人源脂肪酸合酶结构可为聚酮合酶的研究奠定基础。[目的] 在酿酒酵母中表达纯化人源脂肪酸合酶蛋白,确定合适的体外纯化条件。[方法] 以酿酒酵母BJ5464为表达载体,构建带有His和Strep双亲和层析标签的重组质粒,诱导表达蛋白后用亲和层析方法获取目标蛋白,并结合凝胶电泳和快速蛋白质液相层析技术,确定合适的蛋白纯化条件。[结果] 成功构建重组表达质粒pxw55-hfas-cSHII, 并在体外纯化得到合适浓度和纯度的人源脂肪酸合酶蛋白,筛选不同缓冲液条件并结合电子显微镜观察结果反馈,确定合适的蛋白体外纯化体系。[结论] 蛋白电镜结构分析需要有高纯度、合适浓度并且形成正确构象的蛋白样品,而人源脂肪酸合酶蛋白纯化体系的建立和纯化条件的确定为其电镜结构分析提供了良好的样品,为人源脂肪酸合酶的结构解析及结构相似但更为复杂的聚酮合酶蛋白解析奠定了良好基础。     

Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.     

Stabilization of Micrococcus lysodeikticus cells towards lysis by lysozyme using glutaraldehyde: application as a novel biospecific ligand for the purification of lysozyme

Micrococcus lysodeiekticus was stabilized against the lytic action of lysozyme by cross-linking with 5% (v/v) glutaraldehyde for 24 h but still retained its ability to bind lysozyme. An immobilized, biospecific ligand was prepared by covalently binding the cells to glutaraldehyde activated amino-Sepharose followed by stabilization of the cells with glutaraldehyde. Lysozyme bound specifically to this column and could be eluted by glycine/NaOH buffer (50 mM, pH 10.0) containing 2 M KCl.     

Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. In this study, BP-MAR, a 1.3-kb upstream fragment of the 5MAR flanking the chicken lysozyme locus, was tested for its effects on integration and expression of transgenes in transgenic rice plants. Using the Agrobacterium-mediated method, we transformed rice with nine different constructs containing seven and six different promoters and coding sequences, respectively. Genomic Southern blot analyses of 357 independent transgenic lines revealed that in the presence of BP-MAR, 57% of the lines contained a single copy of the transgene, whereas in its absence, only 20% of the lines contained a single copy of the transgene. RNA gel-blot and immunoblot experiments demonstrated that in the presence of BP-MAR, transgene expression levels were similar among different lines. These data were in direct contrast to those derived from transgenes expressed in the absence of BP-MAR, which varied markedly with the chromosomal integration site . Thus, it can be concluded that BP-MAR significantly reduces the variability in transgene expression between independent transformants. Moreover, the presence of BP-MAR appears to confer a copy number-dependent increase in transgene expression, although it does not increase expression levels of individual transgenes. These data contrast with results previously obtained with various MARs that increased expression levels of transgene significantly. Therefore, we conclude that the incorporation of BP-MAR sequences into the design of transformation vectors can minimize position effects and regulate transgene expression in a copy number-dependent way.S.-J. Oh, J.S. Jeong, E.-H. Kim, N.R. Yi and S.-I. Yi contributed equally to the paper     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.     

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system.     

Commercial production of avidin from transgenic maize: characterization of transformant,production, processing,extraction and purification

We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces.An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at –29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.     

Parallel, high-throughput purification of recombinant antibodies for in vivo cell assays

We describe a method for high-throughput, parallel purification of secreted proteins to analyse large numbers of protein samples in cell-based assays for the discovery of protein therapeutics. The procedure is generic and capable of 96 parallel purifications and compatible, in both yield and purity, with a wide assay range. By optimising expression and purification steps as well as using novel hardware, in particular a chromatography press capable to purify target proteins from viscous media, we exemplify the process for the generation of single-chain Fv antibody fragments (scFv) and the purification of full-length IgG. The described process can operate robustly with a throughput of over 2,000 samples per month.