Effect of different immobilization media on breakdown of whey proteins by Streptococcus thermophilus

Green gram (Vigna radiata) is considered the chief legume in Pakistan. Thus, current study was conducted to examine the ameliorating effect of phytohormones pre-treatments under salt stress on proteome profile of green gram by sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The soluble green gram seedlings proteins were resolved on 4% stacking and 12% resolving gels. The SDS-PAGE resolved 24 polypeptide bands ranging from 200 to 17kDa. Among these, 12 out of 24 bands of proteins were essentials house-keeping or growth proteins of green grams. While, 120, 114.6, 51.8, 29.1, and 22.8 kDa bands were over-expressed under 50 to 350mM salt with phytohormones treatments. The others 104.5 kDa, 99.8 kDa, 95.3 kDa, 91.0 kDa, 55 kDa, 46 kDa, and 17kDa bands were related to the GAᴣ, IAA, and SA induced tolerance. Overall 120 kDa, 114.6 kDa, 104.5 kDa, 99.8, 95.3 kDa, 51.8 kDa, 29.1 kDa and 22.8kDa bands were first time identified in the current study. The information retrieved from NCBI protein database, the resolved peptides were principally belonging to 7S and 8S vicilin, 2S, 8S, 11S, and 16.5S globulins. It is determined that seed priming with SA enhanced tolerance in green gram by rapidly synthesizing stress alleviating peptides.Key word: Cluster analysis, dendrogram, mungbean, salt stress, SDS-PAGEINTRODUCTIONVarious world-wide health concerning organization recommended the use of high graded plant protein such as legumes to prevent the risk of metabolic disorder (Hou et al., 2019). Legumes are most important protein crop on the earth. Among the legumes, the green gram is the major pulses. Its seeds are rich in superior quality storage protein, which account 85% of the total protein while, another 15% have not been broadly studied (Yi-Shen et al., 2018). The soluble storage protein comprises of 60% globulins, 25% albumin and 15% prolamins. Globulins are further divided into 3.4% basic-type (7S), 7.6% legumin-type (11S), and 89% vicilin-type (8S) (Mendoza et al., 2001; Itoh et al., 2006). Other than proteins, the green gram seeds also contain starch, fiber, phenolic compound, saponins, vitamins, calcium zinc, potassium, folate, magnesium, manganese and very low in fat that made it meager man’s meat (Hou et al., 2019). It is also a good source of green manure and fodder (El-Kafafi et al., 2015). Its root has ability to fix 30 to 50 Kg/ha atmospheric nitrogen in the soil which is essential for maintaining soil fertility (Chadha, 2010). The green gram is the valuable and the major Rabi pulse crop of Pakistan. Its cultivation area in 2016-2017 was about 179,000 hectares with seed yield of 130,000 tones. In comparison during 2017-2018, it was cultivated on 161,800 hectares land with 118,800 tones seed yield (GOP, 2018). One of the reasons of this 9% decrease in both land and productivity is the shortage of irrigated land due to soil salinity. The salinity induce oxidative bust in the mungbean cells, caused by responsive oxygen species (ROS) such as hydrogen peroxide, singlet oxygen, hydroxyl radical and superoxide radical. The ROS create hindrance in various metabolic processes of plant via interacting with macromolecules like proteins (Alharby et al., 2016). However, phytohormones like gibberellic acid (GAᴣ), indole acetic acid (IAA), and salicylic acid (SA) take part in the biosynthesis of salt tolerance proteins under salinity. These salt tolerance proteins acclimate plants under salinity stress. Application of biotechnology plays a significant role in agriculture (Khan et al., 2017). Therefore, production of particular proteins under salt stress is a specific response of cell which can be analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE is the simple, valid, and cost-effective biochemical marker (Mushtaq et al., 2018). This marker has been widely used to determine the extent of evolutionary variations in crops (El-Kafafi et al., 2015).OBJECTIVES The present study was directed first time with the aim to investigate the toxic effect of sodium chloride (0-350 mM) and stress acclimation by pre-treatment of GAᴣ, IAA, and SA on the proteome profile of NM-92 cultivar of a Pakistani green gram.MATERIALS AND METHODSThe present study was replicated thrice in the plant laboratory of Department of Genetics, Faculty of Science, and University of Karachi. The seeds of mung bean cultivar NM-92 were acquired from National Agricultural Research Centre (NARC), Islamabad. These freshly collected 15 seedsˉ1 treatment / replication were divided into two sets. The first was named as sodium chloride (SC) stress treatments were imbibed in sterile distilled water (DW) whereas, second set soaked in gibberellic acid (GAᴣ) (BDH Chemicals, England), indole acetic acid (IAA) (Fluka, Switzerland), and salicylic acid (SA) (J.T. Baker, Holland) in the separate beaker for 24 hours under dark condition. After 24 hours, given ample time to both the sets at room temperature. After recovery, all 20 treatments were sown in the 150 X 30 mm sized petri-dishes containing 0, 50, 150, 250, and 350 millimolar (mM) sodium chloride solution (Fisher Scientific, UK) for 72 hours.Protein extraction: Protein extraction was done by taking 0.3g of seedlings in an ice chilled mortar and crushed by adding 600µL 0.2 M Tris-HCl buffer having pH 7.5 contained 5% SDS (w/v) and 5% 2-mercaptoethanol (v/v). The homogenate was incubated at 0oC for 30 min., boiled in the water bath for 3 min. at 100oC. Samples were centrifuged in Heraeus Biofuge D-37520, Germany for 30 min. at 8000 rpm. The protein supernatant was saved at below 0°C for quantitative and qualitative determination with minor modifications. The total soluble protein content of the samples was estimated via “Bovine Serum Albumin (BSA) standard curve” and explicit in µg protein milligramˉ1 fresh weight of mung seedlings.Bovine serum albumin standard curve (2000 μg/mL): Total protein standard curve was made by dissolving 0.05g of Bovine Serum Albumin (BSA) in 25mL of distilled water. Ten serial dilutions were made from 0.1 mL to 1mL by BSA solution then performed Lowry. A standard curve of total proteins was plotted by taking BSA absorbance at Y-axis and 2000 μg BSA / mL at X-axisSample preparation for SDS-PAGE: For qualitative assessment of total proteins; the 35μL of saved protein supernatant was combined with 15μL of sample diluting buffer (SDB). The SDB was made up of 0.0625 M Tris-HCl pH 6.8 with 2% of SDS, 10% of glycerol, 0.003% of bromophenol blue dye and 5% of 2-mercaptoethanol. Boil the 50μL protein SDB supernatant at 100oC in water bath for 3 min., centrifuged at 6000 rpm for 4 min. The supernatant was loaded on SDS-PAGE gel with the given formulae. The SDS- PAGE: Total proteins were fractionated via SDS-PAGE with 4% stacking and 12% resolving gel. The resolving gel of 12% was made by taking 6mL solution A, 1.8 mL 3 M Tris 1 M HCl buffer pH 8.8, 144μL 10% SDS, 5.74 mL sterile distilled water, 720μL 1.5% ammonium persulphate (APS) in deionized water and 10μL TEMED. While, stacking was composed of 1.25mL of solution A, 2.5mL of 0.5M Tris 1M HCl buffer pH 6.8, 100μL 10% SDS, 1.8 mL of distilled water, 500μL 1.5% APS and 12μL TEMED. Solution A was prepared by conjoining 30% acrylamide and 0.8% N, N’-methylene-bisacrylamide in deionized water. To avoid polymerization in the beaker; the prepared solution was quickly poured into the 3 mm thick gel plates after adding TEMED. The stacking was lined over resolving gel, then combs were inserted between the gel plates of SCIE-PLAS TV-100 separation system, UK, and allowed to polymerize for ½ an hour. After polymerization gel was placed in the tank which were filled with Tris-Glycine buffer (electrode buffer) pH 8.4 then combs were removed. The electrode buffer contained 0.3% Tris, 1.41% Glycine and 0.1% SDS in 2000mL d/w. The gel was pre-run for 15 min. at 60 volts and 120 mA currents. The prepared SDS-PAGE samples were loaded in wells with BlueStepTM Broad Range Protein Marker, AMRESCO, USA as standard and run at 60 volts & 120 mA for about 45 min. When samples entered in resolving gel, and then gave 100 volts and 200 mA currents for around 2.5 hours. Furthermore, electrophoresis was carried out at a constant watt.The Gel was washed with 30% ethanol on Uni Thermo Shaker NTS-1300 EYELA, Japan at the constant shaking for 30 min. Then gels were placed in 10% glacial acetic acid in 50% methanol solution (Fixative) for 24 hours. SDS Gel was stained until protein bands were visible thereat placed as 5% of Methanol in 7.5% acetic acid glacial solution to destain the bands background. SDS-PAGE stain composed of 0.125% coomassie brilliant blue R-250 dissolved in 40% of Methanol and 7% acetic acid glacial solution. The stain was stirred on Magnetic stirrer & hot plate M6/1, Germany for 6-10 hours before used. Photographs were taken by Sanyo digital camera VPC-T1284BL and bands were scored through numbering pattern. Gels preserved in 10% acetic acid solution at 4°C.Interpretation of bands and data analysis: The total soluble protein bands relative mobility calculated by below formulae and Dendrogram was constructed via SPSS v. 20Where,F=(Migrated distance of protein band)/(Migrated distance of dye front)Slop=(Log MW of protein marker lower limit band–log〖MW of protein marker upper limit band )/(RF protein marker lower limit band –RF of protein marker upper limit band)RESULTS:The total soluble proteins extracted from green gram were perceived by SDS-PAGE Blue StepTm broad range biochemical markers. The protein-based marker was used to evaluate the toxic effect of sodium chloride along with pre-treatments of GAᴣ, IAA, and SA on proteome assay. In the current work, seedlings total soluble proteome resolved 24 polypeptide bands ranging from 200 to 17.1 kDa were recognized by using SDS-PAGE. The figure 1 showed Dendrogram assay, which classified the 20 treatments of SC, GAᴣ, IAA and SA into two major cl

Protein banding patterns of seed obtained by sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) or acid Polyacrylamide gel electrophoresis (A-PAGE) were used to distinguish between different tall fescue (Festuca arundinacea Shreb.) grass cultivars. An electrophoretic formula was determined for each cultivar using relative mobility and the number of protein bands. Distinctive band patterns were obtained enabling the cultivars to be identified using either the SDS-PAGE or the A-PAGE method. Banding patterns were independent of locality or year of production of the seed. Seed protein banding patterns can thus be used as a tool for taxonomie classification of tall fescue grass cultivars.

Present work evaluated the protein quality of defatted white sesame flour and the protein isolates obtained from sesame cake through the alkaline extraction at a pH 9.5. The study was conducted at Department of Foods and Nutrition, Post Graduate and Research Center (PGRC), College of Community Science and MFPI - Quality Control Laboratory, Professor Jayashankar Telangana State Agricultural University, Rajendranagar, Hyderabad (India) during 2021-2023. Defatted white sesame cake was subjected to alkaline extraction at pH 9.5 and resultant isolates were evaluated for nutrient composition, scanning electron microscope imaging, amino acid composition and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The obtained data was subjected to one-way analysis of variance. The white sesame protein isolates had a protein content of 93.83%, isolate recovery of 37.00 g/100 g and protein yield of 71.77%. The non-essential amino acids (NEAAs) content of the defatted white sesame flour and white sesame protein isolates ranged between 68.06% to 70.60% of the total protein content, while 29.40% to 31.94% was essential amino acids (EAAs). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of defatted white sesame flour WSF and white sesame protein isolate (WSPI) exhibited protein bands within the molecular weight range of 20 to 62 kDa. The protein isolates derived from sesame seed cake has promising potential for integration into diverse food formulations, offering an avenue to combat protein deficiencies.

Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS)

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

ABSTRACTNative glutenin aggregates of two different quality flours containing the same high molecular weight (HMW) glutenin subunit compositions were investigated by multistacking sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) procedures. Five stacking gels (4, 6, 8, 10, and 12%) with a separating (resolving) gel of 14% were used to separate nonreduced glutenin aggregates solubilized from flour by SDS sodium phosphate buffer. There were large differences in protein solubility of the two flours. It took 8 hr to extract 91% of total proteins from the strong flour (variety Len) while it took only 2 hr for the weak flour sample 205. Total glutenin proteins and the proportions of glutenins at the different origins (including the origin of the 14% separating gel) were quantified by high‐resolution densitometry procedures. As the duration of extraction increased, both total glutenins and glutenins at the 4% origins increased. The good quality flour Len had a higher total glutenin protein (3% more) and higher proportion of glutenins with the largest molecular sizes (also 3% more) at the 4% origins than the poor quality flour sample 205. After glutenin aggregates from each origin were reduced and analyzed by SDS‐PAGE, the largest glutenins at the 4% origin contained twice the amount of total HMW glutenin subunits when compared to the smaller aggregates at the 12% origin. Among the total HMW glutenin subunits, the proportion of subunit 5 (which published literature reports to be the largest molecular weight based on calculations of DNA‐derived amino acid sequence analysis) was twice that at the 12% origin. A randomized structure of native glutenins is proposed based on the results of our investigations.

Gene Targeting of Mutant COL1A2 Alleles in Mesenchymal Stem Cells From Individuals With Osteogenesis Imperfecta

Breast cancer is one of the most common cancers in women, and rated the second most common cancer and a significant cause of death in females in the Sudan. This study aims to identify tumor protein that elicits humoral immune responses in breast cancer patient in comparison to tissues from healthy individuals as well as from normal tissues of the cancer patient. Serum samples and breast cancer tissue specimens were collected from breast cancer patients (n = 9) and from healthy individuals (n = 5) at Khartoum Teaching Hospital. Breast cancer tissues were homogenized in PBS, centrifuged and the supernatants were lysed in 2X SDS-PAGE sample buffer. The preparation then boiled and the resulting supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Total proteins were separated on SDS-PAGE and transferred to the nitrocellulose paper, then analyzed by immunoblotting for total proteins and serum antibodies using serum from patients with breast cancer and from healthy individuals to enhance humoral immune responses. The SDS-PAGE analysis showed an increase in size of protein bands in the normal control tissues than from breast cancer patients. The Western blot analysis of breast cancer tissues with the serum from breast cancer patients specifically detected major bands than in the normal tissues from the healthy tissues of cancer patients or from healthy individuals. Beside the major bands, additional bands have been detected in breast cancer tissues with the serum from breast cancer patients. The reactive auto-antibodies in patient’s tissues bound to the circulating tumor antigen in patient’s serum and immune complexes would result by Western blotting indication of strong immune response to these proteins. The present study demonstrated that there was a clear decline in the expression of some proteins among breast cancer patients which has been confirmed by strong immune reactions in the Western blotting analysis. Key words: breast cancer , protein expression, SDS-PAGE, Commasie brilliant blue, Western blot.

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

Overcoming in vitro gastric destabilisation of emulsion droplets using emulsion microgel particles for targeted intestinal release of fatty acids

Four strains of eri, Samia cynthia ricini Lepidoptera: Saturniidae that can be identified morphologically and maintained at North East Institute of Science and Technology, Jorhat were characterized based on their protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and DNA by random amplified polymorphic DNA (RAPD) technique. Fiber yield was highest in Gs strain. SDS-PAGE profile showed 11 prominent bands in the strains with molecular weight ranging from 35 to 200 kDa together with 28 minor bands with molecular weight ranging from 33 to 210 kDa. Two polypeptides of molecular weight 90 and 110 kDa were absent in both Y and Ys. Eight random primers and one universal primer used for RAPD analysis generated a total of 79 bands, of which 49 were polymorphic. In both SDS-PAGE and RAPD, the UPGMA based dendrogram showed two clusters: cluster 1 included Gs and G, whereas Y and Ys was grouped in cluster 2 by SDS-PAGE analysis but RAPD analysis grouped Ys and G in cluster 1 and Gs and Y in cluster 2. The range of genetic diversity observed among the strains affirms the potentiality of RAPD technique for identification and selection of distant parents for silkworm hybridization for high silk yield. Key words: Genetic variation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), random amplified polymorphic DNA (RAPD), silkworm, cocoon.

Four strains of eri, Samia cynthia ricini Lepidoptera: Saturniidae that can be identified morphologically and maintained at North East Institute of Science and Technology, Jorhat were characterized based on their protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and DNA by random amplified polymorphic DNA (RAPD) technique. Fiber yield was highest in Gs strain. SDS-PAGE profile showed 11 prominent bands in the strains with molecular weight ranging from 35 to 200 kDa together with 28 minor bands with molecular weight ranging from 33 to 210 kDa. Two polypeptides of molecular weight 90 and 110 kDa were absent in both Y and Ys. Eight random primers and one universal primer used for RAPD analysis generated a total of 79 bands, of which 49 were polymorphic. In both SDS-PAGE and RAPD, the UPGMA based dendrogram showed two clusters: cluster 1 included Gs and G, whereas Y and Ys was grouped in cluster 2 by SDS-PAGE analysis but RAPD analysis grouped Ys and G in cluster 1 and Gs and Y in cluster 2. The range of genetic diversity observed among the strains affirms the potentiality of RAPD technique for identification and selection of distant parents for silkworm hybridization for high silk yield. Key words: Genetic variation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), random amplified polymorphic DNA (RAPD), silkworm, cocoon.

The effects of ionic strength, pH and milk mineral composition on the adsorption of caseins and whey proteins on to particles of hydroxyapatite (HA) were studied by determining the amounts of adsorbed proteins on HA using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and by zeta-potential measurements. The amount of adsorbed proteins increased with increasing ionic strength from 0 to 0.1 M and decreasing pH from 8 to 6, for both sodium caseinate (SC) and whey protein isolate (WPI). In general, when the absolute value of the zeta-potential decreased because of changes in pH or ionic strength, more protein was found to adsorb on to the HA particles. This may be attributed to a decrease in the electrostatic repulsions between the HA particles and the protein species. The effects of the composition of the milk serum were investigated using simulated milk ultrafiltrate (SMUF). Both caseins and whey proteins adsorbed less on to HA particles in SMUF than on to HA particles in water. However, the effect of the composition of SMUF on the adsorption was less pronounced for caseins than for whey proteins. This may be related to the phosphorylated nature of the caseins. As the phosphate groups of the caseins bind more strongly to the HA binding sites than the carboxyl groups of the whey proteins, caseins might compete better with the phosphate and citrate ions in the milk serum for adsorption.

Whey protein and inulin at various weight ratios were dry heated at 60 °C for 5 days under relative humidity of 63%. The heated mixtures were found to have significant changes in browning intensity and zeta-potential compared to untreated mixture. Heated samples showed significantly lower surface hydrophobicity than untreated mixtures. Compared with untreated samples, dry-heated samples showed significantly higher 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) scavenging ability with whey protein to inulin mass ratios of 1:2 and 1:3 and significantly higher 2,2′-Azinobis(2-Ethylbenzothiazoline-6-Sulfonate) (ABTS) scavenging abilities and oxygen radical absorbance capacity (ORAC) at all weight ratios. Dry heat-induced interactions between whey protein and inulin was confirmed by changes in Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profile, Fourier Transform Infrared Spectroscopy (FT-IR) and Far-ultraviolet Circular Dichroism (Far-UV CD) spectra. Dry heating caused physicochemical and structural changes of whey protein and therefore the complexes can be used to improve the antioxidative properties of the mixture under certain conditions.

The overexpression of epidermal growth factor receptor (EGFR) could result in the development of solid tumors of prostate, breast, gastric, colorectal, ovarian, and head and neck, leading to carcinoma. Antibody therapies are ideal methods to overcome malignant diseases. However, immunoribonucleases are a new generation of antibodies in which an RNase binds to a specific antibody and shows a stronger ability to terminate cancer cells. In this study, we engineered Rana pipiens RNase to bind to the scFv of human antiepidermal growth factor receptor antibody. The molecular dynamic simulations confirmed protein stability and the ability of scFv-ranpirnase (rantoxin) to bind to epidermal growth factor receptor protein. Then, the rantoxin construct was synthesized in a pCDNA 3.1 Neo vector. CHO-K1 cells were used as expression hosts and the construct was transfected. Cells were selected by antibiotic therapies using neomycin, 120 mg/ml, and the high-yield colony was screened by real-time polymerase chain reaction (PCR) methods. Then, the recombinant protein production was confirmed using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. The molecular dynamicsimulation (MDS) confirmed that the I467, S468, Q408, and H409 amino acids of EGFR bonded well to rantoxin. As revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses,the rantoxin production and PCRanalysis showed that the T3 colony can produce rantoxin messenger RNA fourfold higher than theGAPDH gene. The immunotoxin function was assessed in A431 cancer cells and EGFR-negative HEK293 cells, and IC50 values were estimated to be 22.4 ± 3 and >620.4 ± 5 nM, respectively. The results indicated that the immunotoxins produced in this study had the potential for use as anticancer drugs.