One-Step Butadiene Purification in a Sulfonate-Functionalized Metal-Organic Framework through Synergistic Separation Mechanism.

Porous materials that could recognize specific molecules from complex mixtures are of great potential in improving the current energy-intensive multistep separation processes. However, due to the highly similar structures and properties of the mixtures, the design of desired porous materials remains challenging. Herein, a sulfonate-functionalized metal-organic framework ZU-609 with suitable pore size and pore chemistry is designed for 1,3-butadiene (C4H6) purification from complex C4 mixtures. The sulfonate anions decorated in the channel achieve selective recognition of C4H6 from other C4 olefins with subtle polarity differences through C-H⋅⋅⋅O-S interactions, affording recorded C4H6/trans-2-C4H8 selectivity (4.4). Meanwhile, the shrunken mouth of the channel with a suitable pore size (4.6 Å) exhibits exclusion effect to the larger molecules cis-2-C4H8, iso-C4H8, n-C4H10 and iso-C4H10. Benefiting from the moderate C4 olefins binding affinity exhibited by sulfonate anions, the adsorbed C4H6 could be easily regenerated near ambient conditions. Polymer-grade 1,3-butadiene (99.5 %) is firstly obtained from 7-component C4 mixtures via one adsorption-desorption cycle. The work demonstrates the great potential of synergistic recognition of size-sieving and thermodynamically equilibrium in dealing with complex mixtures.

Streamlined purification and characterization of Pyrococcus furiosus rubredoxins with different N-terminal modifications by reversed-phase HPLC.

Rubredoxins (Rds), like those from Pyrococcus furious (Pf), have largely been found to be expressed in Escherichia coli (E. coli) as a mixture of different N-terminal forms, which may affect the properties of the protein. The typical procedures for the purification of Rds are cumbersome and usually with low yield. We present herein a streamlined purification strategy based on the reversed-phase high performance liquid chromatography (RP-HPLC), which offers high yield and high resolution after simply one-step purification following pre-treatment. We also show that RP-HPLC can be a valuable tool to gain information related to the thermal decomposition pathway of Pf-Rds.

One-step Ethylene Purification from an Acetylene/Ethylene/Ethane Ternary Mixture by Cyclopentadiene Cobalt-Functionalized Metal-Organic Frameworks.

The separation of ethylene (C2 H4 ) from a mixture of ethane (C2 H6 ), ethylene (C2 H4 ), and acetylene (C2 H2 ) at normal temperature and pressure is a significant challenge. The sieving effect of pores is powerless due to the similar molecular size and kinetic diameter of these molecules. We report a new modification method based on a stable ftw topological Zr-MOF platform (MOF-525). Introduction of a cyclopentadiene cobalt functional group led to new ftw-type MOFs materials (UPC-612 and UPC-613), which increase the host-guest interaction and achieve efficient ethylene purification from the mixture of hydrocarbon gases. The high performance of UPC-612 and UPC-613 for C2 H2 /C2 H4 /C2 H6 separation has been verified by gas sorption isotherms, density functional theory (DFT), and experimentally determined breakthrough curves. This work provides a one-step separation of the ternary gas mixture and can further serve as a blueprint for the design and construction of function-oriented porous structures for such applications.

A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins.

We have developed a new ligation independent cloning (LIC) vector - pSrtA9, which can be utilized for one-step purification of recombinant proteins. The new LIC site in the pSrtA9 vector, hosts a DNA sequence centered on a SfoI restriction site and integrates a coding sequence for sortase A (SrtA) recognition. Preceding the LIC site, pSrtA9 incorporates an N-terminal 6xHis-tag and the catalytic core of SrtA from Staphylococcus aureus (SrtAΔ59). Thus, after cloning and protein expression in Escherichia coli, the resultant fusion protein comprises an N-terminal 6xHis-tag, SrtAΔ59, an L-P-E-T-G linker and the protein of interest at the C-terminus. The fusion protein can be captured onto immobilized Ni-NTA resin and any unwanted proteolysis activity of SrtA is suppressed during the purification by optimisation of solution conditions. Upon addition of Ca2+ and triglycine (Gly3), the immobilized fusion protein undergoes on-column SrtA-mediated cleavage at the T-G bond of LPETG linker to selectively release 90% of the protein of interest within 3 h when incubated at room temperature. This new pSrtA9 vector, thus, offers an efficient method for LIC of genes and a one-step purification procedure to obtain a tag-free recombinant protein, and is therefore suitable for the high-throughput proteins production.

A highly efficient, one-step purification of the Hsp70 chaperone Ssa1.

Chaperone proteins are required to maintain the overall fold and function of proteins in the cell. As part of the Hsp70 family, Ssa1 acts to maintain cellular proteostasis through a variety of diverse pathways aimed to preserve the native conformation of target proteins, thereby preventing aggregation and future states of cellular toxicity. Studying the structural dynamics of Ssa1 in vitro is essential to determining their precise mechanisms and requires the development of purification methods that result in highly pure chaperones. Current methods of expressing and purifying Ssa1 utilize affinity tagged constructs expressed in Escherichia coli, however, expression in an exogenous source produces proteins that lack post-translational modifications leading to undesired structural and functional effects. Current protocols to purify Ssa1 from Saccharomyces cerevisiae require large amounts of starting material, multiple steps of chromatography, and result in low yield. Our objective was to establish a small-scale purification of Ssa1 expressed from its endogenous source, Saccharomyces cerevisiae, with significant yield and purity. We utilized a protein A affinity tag that was previously used to purify large protein complexes from yeast, combined with magnetic Dynabeads that are conjugated with rabbit immunoglobulin G (IgG). Our results show that we can produce native, highly pure, active Ssa1 via this one-step purification with minimal amounts of starting material, and this Ssa1-protein A fusion does not alter cellular phenotypes. This methodology is a significant improvement in Ssa1 purification and will facilitate future experiments that will elucidate the biochemical and biophysical properties of Hsp70 chaperones.

One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins.

BACKGROUND: Bacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Fusion to highly soluble carrier proteins such as Maltose Binding Protein (MBP) often improves their folding and solubility, but self-association may still occur. For instance, HPV E6 oncoproteins, when produced as MBP-E6 fusions, are expressed as mixtures of biologically inactive oligomers and active monomers. While a protocol was previously developed to isolate MBP-E6 monomers for structural studies, it allows the purification of only one MBP-E6 construct at the time. Here, we explored a parallelizable strategy more adapted for biophysical assays aiming at comparing different E6 proteins. RESULTS: In this study, we took advantage of the distinct size and diffusion properties of MBP-E6 monomers and oligomers to separate these two species using a rapid batch preparation protocol on affinity resins. We optimized resin reticulation, contact time and elution method in order to maximize the proportion of monomeric MBP-E6 in the final sample. Analytical size-exclusion chromatography was used to quantify the different protein species after purification. Thus, we developed a rapid, single-step protocol for the parallel purification of highly monomeric MBP-E6 samples. MBP-fused HPV16 E6 samples obtained by this approach were validated by testing the binding to their prototypical peptide targets (the LXXLL motif from ubiquitine ligase E6AP) by BIAcore-SPR assay. CONCLUSIONS: We have designed a rapid single-step batch affinity purification approach to isolate biologically active monomers of MBP-fused E6 proteins. This protocol should be generalizable to isolate the monomer (or the minimal biologically active oligomer) of other proteins prone to self-association.

Enhancing recombinant interleukin-6 production yield by fermentation optimization, two-step denaturing, and one-step purification.

Interleukin-6 a pleiotropic cytokine involved in a wide range of biological activities. So the large-scale production of biologically active recombinant human interleukin-6 is important for its structural and functional studies. Here, we report an optimized method for shake flask fermentation and a simplified high-yield purification procedure for the recombinant interleukin-6. This high-yield expression method not only involves the optimization of the fermentation condition but also the single step purification method as well as a two-step denaturing and one-step refolding process. This approach replaces the more conventional procedure of protein solubilization and refolding. Through applying these strategies, the final cell density and overall product yield of the recombinant human interleukin-6 were obtained as 20.4 g as cell biomass and 150 mg as purified active protein from the I-L of the culture. The purified protein was characterized by HPLC and SDS-PAGE. The results of the current work demonstrate that the described method may be used to develop the process for industrial-scale production of the biologically active recombinant interleukin-6 protein.

Evaluating the autoinduction expression system and one-step purification for high-level expression and purification of gallbladder-derived rhIL-1Ra.

Recent advancement in fermentation technologies resulted in the increased yields of recombinant proteins of biopharmaceutical and medicinal importance. Consequently, there is an important task to develop simple and easily scalable methods that can facilitate the production of high-quality recombinant protein. Most of the recent reports described the expression of recombinant human IL-1 receptor antagonist (rhIL-1Ra) in Escherichia coli using isopropyl-ß-d-thiogalacto pyranoside (IPTG), a nonmetabolizable and expensive compound, as an expression inducer. In this study, we describe the expression and one-step purification of gallbladder-derived rhIL-1Ra by autoinduction in E. coli. This method includes special media that automatically induce the target protein expression from T7 promoter and allow the production of the target protein in high yield than the conventional IPTG induction method. In addition to fermentation process improvements, one-step purification strategy is essential to make the process economical. We developed a single-step cation exchange chromatography and obtained 300 mg/L of rhIL-1Ra with 98% purity. Purified protein was characterized by SDS-PAGE and Ion exchange HPLC (IEX-HPLC). The described method can be used to scale up the production of rhIL-1Ra and other recombinant proteins.

High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli.

We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.

Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN.

Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-ß-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single-step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost.

Heterofunctional Magnetic Metal-Chelate-Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity.

In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly-His-tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor-made magnetic chelate-epoxy supports. In order to selectively adsorb and then covalently immobilize the poly-His-tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co(2+)-chelate groups (38 µmol Co(2+)/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine-tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free-enzyme-catalyzed reaction. The enantiomeric excess (ee) of (R)-benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles.

A Simple and Cost-Efficient Platform for a Novel Porcine Circovirus Type 2d (PCV2d) Vaccine Manufacturing.

Porcine circovirus type 2d (PCV2d) is becoming the predominant PCV genotype and considerably affects the global pig industry. Nevertheless, currently, no commercial PCV2d vaccine is available. Preventing and controlling the disease caused by PCV2d is therefore based on other genotype-based vaccines. However, their production platforms are laborious, limited in expression level, and relatively expensive for veterinary applications. To address these challenges, we have developed a simple and cost-efficient platform for a novel PCV2d vaccine production process, using fed-batch E. coli fermentation followed by cell disruption and filtration, and a single purification step via cation exchange chromatography. The process was developed at bench scale and then pilot scale, where the PCV2d subunit protein yield was approximately 0.93 g/L fermentation volume in a short production time. Moreover, we have successfully implemented this production process at two different sites, in Southeast Asia and Europe. This demonstrates transferability and the high potential for successful industrial production.

Linker-peptide-mediated one-step purification and immobilization of α-L-rhamnosidase from Bacteroides thetaiotaomicron for direct biotransformation from epimedin C to icariin.

Icariin, the most effective bioactive component in Epimedium, is also the index component of Epimedium quality control in Pharmacopoeia. It was a very attractive approach for bioconversion from epimedin C to icariin. However, its potential was impeded by poor stability and non-recyclable properties of free enzymes. In this study, we have fused the linker (4LP) to α-L-rhamnosidase BtRha and successfully prepared the immobilized enzyme (incubated 4LP-BtRha@Na-Y) to produce icariin from epimedin C. The activity recovery of 4LP-BtRha@Na-Y was 79.6 %, and enzyme activity was 209.8 U/g, which was 1.75-fold and 1.6-fold higher than that of immobilized BtRha (BtRha@Na-Y), respectively. The optimal reaction temperature and pH of 4LP-BtRha@Na-Y was 55 °C and 6.5, respectively. The thermal stability of immobilized enzyme was significantly improved by incubation in phosphate buffer containing 20 % glycerol and 10 % fructose. The kcat/Km value of incubated 4LP-BtRha@Na-Y was 7.98 × 105 s-1M-1, which increased by 8 % compared with free BtRha. Finally, under suitable conditions, 1 g/L epimedin C was transformed into icariin with icariin yield 75.1 %, and the relative conversion rate retained 74.9 % after reused 13 cycles. This experiment provides a new idea for one-step purification and immobilization of α-L-rhamnosidase for direct biotransformation from epimedin C to icariin, which will have great prospects in food and pharmaceutical production.

[Determination of 14 ß-agonists in animal meat by ultra high performance liquid chromatography-tandem mass spectrometry].

The addition of ß-agonists to animal feed can significantly improve the lean-meat rate of pigs, cattle, sheep, and other animals. However, the food residues of ß-agonists are harmful to human health. When meat with ß-agonist residues is consumed, poisoning symptoms such as palpitation, dizziness, and muscle tremors may develop, and damage to the cardiovascular system, liver, and kidney may occur. In this study, a method based on ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established for the rapid detection of 14 ß-agonists (clenbuterol, salbutamol, ractopamine, clorprenaline, terbutaline, tulobuterol, bromobuterol, bambuterol, zilpaterol, mabuterol, fenoterol, arformoterol, cimaterol, and cimbuterol) in animal food sources. The sample pretreatment method and chromatographic conditions were optimized. The samples were hydrolyzed with ß-glucuronidase hydrochloride/aryl sulfate esterase in ammonium acetate buffer (pH 5.2). Enzymatic hydrolysis was performed in a constant-temperature water bath ((36±2) ℃) oscillator for 16 h. The samples were cooled to room temperature and extracted with 0.5% formic acid acetonitrile. NaCl was added to separate the organic and aqueous phases, and 5 mL of the upper organic layer was purified using a one-step purification solid-phase extraction column. After drying with nitrogen at 50 ℃, the residue was dissolved in 0.4 mL of 0.2% formic acid aqueous solution. The samples were passed through a 0.22 µm filter and detected by UHPLC-MS/MS with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The analytes were separated on a Phenomenex Kinetex F5 column and detected by positive-ion scanning in multiple-reaction monitoring (MRM) mode. Internal and external standard methods were used for quantitative analysis. The effects of the extract pH, solid-phase extraction column, purification method, and dissolved solution on the extraction efficiency were optimized during pretreatment. UHPLC-quadrupole time-of-flight MS was used to verify the purification effect of the one-step purification solid-phase extraction column, and the results indicated that this type of column could remove most of the phospholipids, sphingolipids, and glycerides in the sample extract. The factors influencing the different chromatographic columns and mobile phases were investigated. MS scanning was conducted in positive-ion mode with needle pump injection in mass-only mode, and the two daughter ions with the highest responses for each target were selected as the quantitative and qualitative ions. The declustering potential (DP) and collision energy (CE) of each ion were separately optimized in MRM mode. The switching mode of the mass spectrum and waste liquid was used, and the mobile phase was switched to waste liquid after all the target peaks were removed. These steps ensured that impurities in the sample flowed out of the column in a timely manner and that the effects of excessive impurities on the mass spectra were avoided. The 14 ß-agonists showed good linear relationships in the range of 1.0-50 µg/L, with correlation coefficients of >0.99. The limits of detection (LODs) and quantification (LOQs) were in the range of 0.1-0.2 and 0.3-0.6 µg/kg, respectively. The average recoveries of the 14 ß-agonists ranged from 70.25% to 117.48%, with relative standard deviations (RSDs) in the range of 0.63%-14.29% at low, medium, and high spiked levels. Pork, beef, and mutton samples were selected and analyzed using the developed method. The results were close to those of the national standard method, indicating that the method is accurate and reliable. Moreover, the proposed method has good stability and high accuracy; thus, it is suitable for the qualitative and quantitative determination of ß-agonists in animal meat.

An easy pipeline for one-step purification of SARS-CoV-2 nucleocapsid protein from insect cell suspension culture.

The COVID-19 pandemic has demanded a range of biotechnological products for detection of SARS-CoV-2 variants and evaluation of human seroconversion after infection or vaccination. In this work, we describe an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used for infection of lepidopteran Sf9 cells adapted to high-density suspension. Using Tris-HCl pH 8.0 buffer as mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg N protein could be purified from each 50 mg of total protein from clarified supernatant. Such protein amount allows the manufacturing of serological tests and the development of basic studies on cellular responses to SARS-CoV-2.

One-Step Generation and Purification of Cell-Encapsulated Hydrogel Microsphere With an Easily Assembled Microfluidic Device.

Cell-laden hydrogel microspheres with uniform size show great potential for tissue repair and drug screening applications. Droplet microfluidic systems have been widely used for the generation of cell-laden hydrogel microspheres. However, existing droplet microfluidic systems are mostly based on complex chips and are not compatible with well culture plates. Moreover, microspheres produced by droplet microfluidics need demulsification and purification from oil, which requires time and effort and may compromise cell viability. Herein, we present a simple one-step approach for producing and purifying hydrogel microspheres with an easily assembled microfluidic device. Droplets were generated and solidified in the device tubing. The obtained hydrogel microspheres were then transferred to a tissue culture plate filled with cell culture media and demulsified through evaporation of the oil at 37°C. The removal of oil caused the gelled microspheres to be released into the cell culture media. The encapsulated cells demonstrated good viability and grew into tumor spheroids in 12-14 days. Single cell-laden hydrogel microspheres were also obtained and grown into spheroid in 14 days. This one-step microsphere generation method shows good potential for applications in automated spheroid and organoid cultures as well as drug screening, and could potentially offer benefits for translation of cell/microgel technologies.

Development and validation of the one-step purification method coupled to LC-MS/MS for simultaneous determination of four aflatoxins in fermented tea.

Aflatoxin B1 is the potential chemical contaminant of most concern during the production and storage of fermented tea. In this work, a simple, fast, sensitive, accurate, and inexpensive method has been developed and validated for the simultaneous detection of four aflatoxins in fermented tea based on a modified sample pretreatment method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aflatoxins were extracted using acetonitrile and purified using mixed fillers (carboxyl multiwalled carbon nanotubes, hydrophilic-lipophilic balance, silica gel). Under optimum LC-MS conditions, the limits of quantification (LOQs) were 0.02-0.5 µg·kg-1. Recoveries from aflatoxins-fortified tea samples (1-12 µg·kg-1) were in the range of 78.94-105.23% with relative standard deviations (RSDs) less than 18.20%. The proposed method was applied successfully to determine aflatoxin levels in fermented tea samples.

Engineering of a chitosanase fused to a carbohydrate-binding module for continuous production of desirable chitooligosaccharides.

Chitooligosaccharides (CHOS) with multiple biological activities are usually produced through enzymatic hydrolysis of chitosan or chitin. However, purification and recycling of the enzyme have largely limited the advancement of CHOS bioproduction. Here, we engineered a novel enzyme by fusing the native chitosanase Csn75 with a carbohydrate-binding module (CBM) that can specifically bind to curdlan. The recombinase Csn75-CBM was successfully expressed by Pichia pastoris and allowed one-step purification and immobilization in the chitosanase immobilized curdlan packed-bed reactor (CICPR), where a maximum adsorption capacity of 39.59 mg enzyme/g curdlan was achieved. CHOS with degrees of polymerization of 2-5 (a hydrolysis yield of 97.75%), 3-6 (75.45%), and 3-7 (73.2%) were continuously produced by adjusting the ratio of enzyme and chitosan or the flow rate of chitosan. Moreover, the CICPR exhibited good stability and reusability after several cycles. The recombinase Csn75-CBM has greatly improved the efficiency of the bioproduction of CHOS.

Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System.

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137-195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.