Effective detection of proteins following electrophoresis using extracts of locally available food species

Procedures in life sciences research laboratories often require chemicals and plasticware that are costly, toxic or pose a risk to the environment. Therefore, sustainable alternatives would be of interest, provided that they generate suitable data quality. Coomassie blue and silver staining are the most widely used methods for detecting proteins following electrophoresis in the laboratory. However, their use presents challenges in terms of safety and waste management. In the current study, aqueous extracts were prepared from a series of common food species and evaluated as alternative stains for protein detection. Beets, blueberries, purple cabbage, raspberries and strawberries were employed to stain identical proteins separated under the same conditions in electrophoresis gels. Extracts of the first two species resulted in protein bands that were detectable through visible light transillumination, whereas extracts from all five species generated specific protein bands under ultraviolet light. The raspberry-derived extract was selected for further study based on the brightness of the fluorescent protein bands and minimal background staining. For both bovine serum albumin and lysozyme at 2.5 μg and 0.5 μg protein per band, the mean signal intensities obtained with raspberry extract staining were just below half of those obtained with Coomassie blue. Furthermore, the mean intensities using raspberry extract were equivalent to those obtained using Coomassie blue in the detection of 0.1 μg protein. Therefore, raspberry could be used to produce an effective stain for the routine laboratory analysis of proteins

Type II antifreeze proteins from smelt (Osmerus mordax) and Atlantic herring (Clupea harengus harengus) : similarity to the C-type lectin family

The structural diversity of fish antifreeze proteins is believed to result from their independent origins in separate fish taxonomic groups. Therefore, antifreeze proteins(AFPs) from two fish species distantly related to all others known to produce AFPs were studied in order to gain a better understanding of the diversity and the distribution of the different AFP types. Results of this study suggest that homologous type II fish AFPs have evolved independently in three separate groups offish but that they originate from members of a single protein family, the calcium-dependent (C-type) lectins. Moreover, the homology among the three AFPs and the C-type lectins has allowed the recently determined structure of a C-type lectin domain to serve as a prototype for the type II AFPs. This will provide the basis for determining the functional mechanism of this AFP type. These findings also suggest that study of the AFP variants present in diverse fish species may lead to the further insight into the origins, the structures, and the mechanisms of action of the different fish AFPs. -- AFPs were isolated from the blood plasma of smelt (Osmerus mordax) and Atlantic herring (Clupea harengus harengus) using gel filtration, ion exchange chromatography, and reverse phase HPLC. Characterization of smelt AFP revealed six isoforms, each with a Mr of 24,000. Amino acid analysis and endoglycosidase digestion revealed that smelt AFP contained N-linked carbohydrate. The herring AFP had two isoforms with a Mr of 14,600 and no carbohydrate. Further analysis of these proteins revealed that, like the type II AFP of sea raven (Hemitripterus americanus) they were both cystine-rich and sensitive to sulfhydryl reducing agents. Antisera raised against herring and sea raven AFPs cross-reacted with smelt AFP. However, the thermal hysteresis activities of smelt and herring AFPs were lower than that of sea raven AFP. -- cDNA libraries were constructed from livers of smelt and herring. Libraries were screened using an oligonucleotide with a sequence derived from tryptic peptides of smelt and herring AFPs. AFP cDNA clones were isolated and sequenced and the primary structures of smelt and herring AFPs were deduced. Protein sequence database searches, sequence alignments, and statistical analyses showed that the AFPs of smelt, herring, and sea raven were homologous to one another and to the carbohydrate-recognition domains (CRDs) present in a family of Ca²⁺-dependent (C-type) lectins. Furthermore, all but one of the residues known to define the hydrophobic cores of a C-type lectin CRD were present in these AFPs. One CRD Ca²⁺-binding site appeared conserved in smelt and herring AFPs and these AFPs did require Ca²⁺ for thermal hysteresis activity. Alignment of sea raven AFP with C-type CRDs suggested that it had no functional Ca²⁺-binding sites and the thermal hysteresis activity of this AFP did not require Ca²⁺. -- Affinity chromatography and agglutination experiments showed that, unlike the C-type lectins, the type II AFPs did not appear to bind strongly to carbohydrates. However, a trace of smelt AFP was retarded on a matrix containing galactose suggesting a slight affinity for this carbohydrate. Two proteins in the C-type lectin family were isolated and tested for thermal hysteresis activity. Neither human pancreatic stone protein nor a C-type lectin from the venom of a snake (Crotalus atrox) showed detectable thermal hysteresis activity. -- Together, these results suggest that the type II AFPs of smelt, herring, and sea raven are related and that all three have evolved from the CRDs of preexisting proteins in the C-type lectin family, the type II AFPs have a fold similar to that of the lectins, and some resemble lectins in requiring Ca²⁺ for activity. However, unlike the lectins, the AFPs appear to interact primarily with ice crystals rather than with carbohydrate

Expression of recombinant Atlantic salmon serum C-type lectin in Drosophila melanogaster Schneider 2 cells

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a soluble C-type lectin that binds bacteria, including salmon pathogens. This lectin is a cysteine-rich oligomeric protein. Consequently, a Drosophila melanogaster expression system was evaluated for use in expressing SSL. A cDNA encoding SSL was cloned into a vector designed to express it as a fusion protein with a hexahistidine tag, under the control of the Drosophila methallothionein promoter. The resulting construct was stably transfected into Drosophila S2 cells. After CdCl2 induction, transfected S2 cells secreted recombinant SSL into the cell culture medium. A cell line derived from stably transformed polyclonal cell populations expressing SSL was used for large-scale expression of SSL. Recombinant SSL was purified from the culture medium using a two-step purification scheme involving affinity binding to yeast cells and metal-affinity chromatography. Although yields of SSL were very low, correct folding and functionality of the recombinant SSL purified in this manner was demonstrated by its ability to bind to Aeromonas salmonicida. Therefore, Drosophila S2 cells may be an ideal system for the production of SSL if yields can be increased.Peer reviewed: YesNRC publication: Ye

Characterization of shrimp oil from Pandalus borealis by high performance liquid chromatography and high resolution mass spectrometry

Northern shrimp (Pandalus borealis) oil, which is rich in omega-3 fatty acids, was recovered from the cooking water of shrimp processing facilities. The oil contains significant amounts of omega-3 fatty acids in triglyceride form, along with substantial long-chain monounsaturated fatty acids (MUFAs). It also features natural isomeric forms of astaxanthin, a nutritional carotenoid, which gives the oil a brilliant red color. As part of our efforts in developing value added products from waste streams of the seafood processing industry, we present in this paper a comprehensive characterization of the triacylglycerols (TAGs) and astaxanthin esters that predominate in the shrimp oil by using HPLC-HRMS and MS/MS, as well as 13C-NMR. This approach, in combination with FAME analysis, offers direct characterization of fatty acid molecules in their intact forms, including the distribution of regioisomers in TAGs. The information is important for the standardization and quality control, as well as for differentiation of composition features of shrimp oil, which could be sold as an ingredient in health supplements and functional foods.Peer reviewed: YesNRC publication: Ye