Protein Extraction Methods Research Articles

Extraction of Total Protein from Axillary Buds of Sugarcane (Saccharum spp.) for Proteomic Analysis

Since sugar cane production depends on the germination of axillary buds, the analysis of the products of gene expression in axillary buds from different sugarcane varieties (Saccharum spp.) in each growth cycle is important to reveal the proteins related to sprouting potential for crop renovation and consequent increase in sugarcane production. Current study has employed a simple method for protein extraction from axillary buds of RB867515, RB866928 and CTC04 sugarcane varieties to obtain protein fractions for two-dimensional electrophoresis and proteomic analysis. The TCA/acetone method for protein extraction with modifications in the centrifugation processes and in the incubation time of samples revealed a large number of well-defined bands in SDS-PAGE system with molecular weights ranging between 120 and 10 kDa. The evidence that the method TCA/acetone modified (1) by using 200 mg of tissue axillary buds; (2) by inclusion of an incubation step at room temperature for 20 min under stirring; (3) by aliquots division for a better recovery from the supernatant; (4) by the absence of phenol in one extraction phase; and (5) by the increase in centrifugal rotation from 12,000×g to 13,860×g and an increase from 15 to 30 min centrifugation time (in the precipitation and wash steps) was more efficient than the other methods used for sugarcane, is one more example showing that methods for extracting proteins in plants are specific to different tissues.

Biogas potential of green biomass after protein extraction in an organic biorefinery concept for feed, fuel and fertilizer production

The biogas potential of the residual fractions of four organically grown green crops after protein extraction was studied. The protein extraction method involved screw pressing of freshly harvested biomass to obtain a plant juice, followed by precipitation of the proteins. After protein extraction, 95% of organic matter was still present in the residual press cake and juice. Methane yields in the range of 219–375 and 429–539 ml-CH4 g-VS−1 were obtained for the mono-digestion of press cake and the residual juice, respectively, and up to 81% of the methane potential of the fresh crops was recovered in the two residual fractions when evaluated separately. Co-digestion of the press cake and the residual juice at the organic matter ratio at which those fractions leave the biorefinery, resulted in a methane yield of 400 ml-CH4 g-VS−1 according to the regression model equation developed for red clover. Consequently, 65% of the methane potential from fresh red clover could be recovered by co-digestion of the residual fractions from the green biorefinery after extraction of proteins.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

Defatting and Sonication Enhances Protein Extraction from Edible Insects.

Edible insects are attracting growing interest as a sustainable source of protein for addition to processed meat and dairy products. The current study investigated the optimal method for protein extraction from mealworm larvae (Tenebrio molitor), cricket adults (Gryllus bimaculatus), and silkworm pupae (Bombyx mori), for use in further applications. After defatting with n-hexane for up to 48 h, sonication was applied for 1-20 min and the protein yield was measured. All samples showed a total residual fat percentage below 1.36%, and a 35% to 94% improvement in protein yield (%). In conclusion, defatting with n-hexane combined with sonication improves the protein yield from insect samples.

A comparison of protein extraction methods optimizing high protein yields from marine algae and cyanobacteria

Algae are a diverse group of aquatic autotrophs that represent important models for studying morphology, physiology, and molecular biology. Many, because of their adaptation to the marine environment, cell wall composition, and secondary metabolites, present difficulties when trying to extract significant amounts of protein for analyses. In this study, we compared protein extraction methods for 39 different species of marine algae and cyanobacteria. Since successful protein detection requires the liberation of protein from a cell, the goal of the current investigation was to establish a high yield protein extraction method that would allow for protein extraction from different organisms ranging from cyanobacteria, unicellular, multicellular, and coenocytic algae. Six protein extraction assays were tested (three from literature citations and three from commercially available kits). A modification of the technique originally described by Barbarino and Lourenco (J Appl Phycol 17:447–460, 2005) displayed significantly higher yields (p > 0.001, average of 6.7–30.87% dry weight) in all algal and cyanobacterial species examined. Protein yields using this modified procedure were especially successful in the Trebouxiophyceae, Bangiophyceae, Phaeophyceae, and Cyanobacteria.

Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample.

BackgroundThe elucidation of complex biological systems requires integration of multiple molecular parameters. Accordingly, high throughput methods like transcriptomics, proteomics, metabolomics and lipidomics have emerged to provide the tools for successful system-wide investigations. Unfortunately, optimized analysis of different compounds requires specific extraction procedures in combination with specific analytical instrumentation. However, the most efficient extraction protocols often only cover a restricted number of compounds due to the different physico-chemical properties of these biological compounds. Consequently, comprehensive analysis of several molecular components like polar primary metabolites next to lipids or proteins require multiple aliquots to enable the specific extraction procedures required to cover these diverse compound classes. This multi-parallel sample handling of different sample aliquots is therefore not only more sample intensive, it also requires more time and effort to obtain the required extracts.ResultsTo circumvent large sample amounts, distributed into several aliquots for the comprehensive extraction of most relevant biological compounds, we developed a simple, robust and reproducible two-phase liquid–liquid extraction protocol. This one-step extraction protocol allows for the analysis of polar-, semi-polar and hydrophobic metabolites, next to insoluble or precipitated compounds, including proteins, starch and plant cell wall components, from a single sample. The method is scalable regarding the used sample amounts but also the employed volumes and can be performed in microcentrifuge tubes, enabling high throughput analysis. The obtained fractions are fully compatible with common analytical methods, including spectroscopic, chromatographic and mass spectrometry-based techniques. To document the utility of the described protocol, we used 25 mg of Arabidopsis thaliana rosette leaves for the generation of multi-omics data sets, covering lipidomics, metabolomics and proteomics. The obtained data allowed us to measure and annotate more than 200 lipid compounds, 100 primary metabolites, 50 secondary metabolites and 2000 proteins.ConclusionsThe described extraction protocol provides a simple and straightforward method for the efficient extraction of lipids, metabolites and proteins from minute amounts of a single sample, enabling the targeted but also untargeted high-throughput analyses of diverse biological tissues and samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0146-2) contains supplementary material, which is available to authorized users.

Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52–63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14–16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry

A critical step in proteomic analyses comprises the implementation of a reliable cell lysis method with high yields of qualitative proteins. In Mycobacteria, the protein extraction step is often hampered by the thick waxy cell wall which is rich in mycolic acids. Harsh disruption techniques to release proteins from the cells are thus required. Here, we demonstrate an optimized protein extraction procedure for Mycobacterium tuberculosis (Mbt) that results in protein extracts that are useful for all currently used proteomics platforms, including gel and LC-MS based strategies. We compared the effectiveness of using both thiourea and urea and/or SDS and DTT in the solubilization buffer, in combination or not with sonication and/or bead beating. After some preliminary optimization steps on fast-growing Mbt-like organisms, namely Mycobacterium smegmatis and Mycobacterium fortuitum, the final protein extraction protocol was tested on M. tuberculosis. Based on the concentrations of the proteins recovered from each of the tested methods and on the quality of the extracted proteins as evaluated by SDS PAGE, we propose a lysis buffer that contains both thiourea and urea, in combination with two mechanical cell disruption methods: sonication and bead beating. The optimized protocol results in protein extracts that are useful in M. tuberculosis proteomics studies based on any proteomics strategy or platform.

Quantitative extracellular matrix proteomics to study mammary and liver tissue microenvironments

Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.

Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue.

Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK.

A protein extraction method for low protein concentration solutions compatible with the proteomic analysis of rubber particles.

The extraction of high-purity proteins from the washing solution (WS) of rubber particles (also termed latex-producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high-purity WS proteins. We improved our current borax and phenol-based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol-based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high-quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP-extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.

Protocol: MYTHBUSTERS: a universal procedure for sample preparation for mass spectrometry.

Improvements in proteomic strategies from the development of new and robust separation and identification techniques have led to broad applications of proteomics to solve numerous biological questions. For all analyses, sample quality is unquestionably a critical factor; therefore protein extraction is of outmost importance. The ideal extraction method should provide reproducible spectra of the most comprehensive repertoire of proteins, while minimizing sample loss and degradation. It is already known that to capture the whole proteome is an unenforceable task. Many protein extraction protocols have been described, yet there is no "one perfect procedure" taking into account the vast diversity of biological and physical properties of proteins, including their charge, size, hydrophobicity, interactions and sub-cellular localization. The research presented here reflects the main obstacle occurring in proteomic experimental design; i.e. the lack of reproducibility as a result of alterations in protein extraction methods. We have performed a series of experiments, aimed towards identification of the aptamer-binding partners in cancerous cells. Aptamers are chemically synthesized, short, single-stranded nucleic acids with a strictly defined three-dimensional structure, which allows them to interact with a target molecule with high affinity. The low immunogenicity and cellular- targeting properties of aptamers might facilitate design of suitable drugs with low side-effects. Aptamers can be used for identification of molecules associated with a pathogenic state of a cell. Aptamers can be considered as a powerful tool, since they possess unique properties to benefit cancer diagnosis, prevention and treatment. We have used different types of protein extraction methods prior to analyses of complex biological samples by mass spectrometry, based on slight changes of homogenization buffers, and have observed the changes in the identified compounds. These results should prove to be very useful for future proteomic studies and the design of studies in terms of sample preparation, especially sample homogenization and protein extraction.

A method for whole protein isolation from human cranial bone

The presence of the dense hydroxyapatite matrix within human bone limits the applicability of conventional protocols for protein extraction. This has hindered the complete and accurate characterization of the human bone proteome thus far, leaving many bone-related disorders poorly understood. We sought to refine an existing method of protein extraction from mouse bone to extract whole proteins of varying molecular weights from human cranial bone. Whole protein was extracted from human cranial suture by mechanically processing samples using a method that limits protein degradation by minimizing heat introduction to proteins. The presence of whole protein was confirmed by western blotting. Mass spectrometry was used to sequence peptides and identify isolated proteins. The data have been deposited to the ProteomeXchange with identifier PXD003215. Extracted proteins were characterized as both intra- and extracellular and had molecular weights ranging from 9.4 to 629 kDa. High correlation scores among suture protein spectral counts support the reproducibility of the method. Ontology analytics revealed proteins of myriad functions including mediators of metabolic processes and cell organelles. These results demonstrate a reproducible method for isolation of whole protein from human cranial bone, representing a large range of molecular weights, origins and functions.

Protein-Based Hydrogels Derived from Industrial Byproducts Containing Collagen, Keratin, Zein and Soy

Proteins are renewable resources rich in animals and plants and can be extracted from the agricultural and breeding industry byproducts and wastes. Collagen, keratin, zein and soy proteins are found in these byproducts and are extensively studied for protein-based hydrogels, which are especially attractive as a promising biomaterial due to their excellent biocompatibility, biodegradability, nontoxicity and low immune response. In recent years, hydrogels have been playing an increasingly important role in tissue engineering, drug delivery, nutrient encapsulation, controlled release of pesticides and other fields. The diversity of the amino acid composition of proteins leads to significant differences in their processing strategies, and the properties of the resultant hydrogels. This review covers the extraction methods for collagen, keratin, zein and soy proteins from various industrial byproducts, the synthesis of hydrogels from these protein sources, as well as their potential applications.

Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations.

Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms.

Cassava: meeting the global protein need

Cassava (Manihot esculenta Crantz) is a nutty flavored, starch-tuber perennial woody shrub originated from tropical America belongs to Euphorbiaceae family of plants. After rice and maize, it is considered as the third largest source of carbohydrate food in the tropics and its sweet, chewy underground tuber is one of the popular edible root-vegetables. It is ranked as a 21st century crop, as it acknowledges to the universal economy trends and climatic changes. Currently, use of cassava leaves as a potential source of protein, vitamins and minerals was reviewed. The effect of malnutrition on health and development of people and its control by using cassava leaves as a protein rich source were briefly discussed. Cascade use of cassava leaves, in industrial applications like natural filler for potential reinforcement of polypropylene based composites was also presented. Although, cassava leaves are vital source of essential nutrients, their anti-nutrients and cyanogenic glucosides content limits their consumption, which can be overcome by the development of an efficient, simple and low-cost processing methods for protein extraction from cassava leaves. There are supporting evidences for efficacy of cassava leaf protein in reducing the effect of malnutrition by the intake of protein rich cassava leaves, fortified with various common food items. So consumption of cassava leaves enriched with high protein, vitamin and mineral contents with the development of suitable processing technology to remove anti-nutrients can be an alternative source to meet the global protein demand.

Understanding leaf membrane protein extraction to develop a food-grade process

Leaf membrane proteins are an underutilised protein fraction for food applications. Proteins from leaves can contribute to a more complete use of resources and help to meet the increasing protein demand. Leaf protein extraction and purification is applied by other disciplines, such as proteomics. Therefore, this study analysed proteomic extraction methods for membrane proteins as an inspiration for a food-grade alternative process. Sugar beet leaves were extracted with two proteomic protocols: solvent extraction and Triton X-114 phase partitioning method. Extraction steps contributed to protein purity and/or to selective fractionation, enabling the purification of specific proteins. It was observed that membrane proteins distributed among different solvents, buffers and solutions used due to their physicochemical heterogeneity. This heterogeneity does not allow a total membrane protein extraction by a unique method or even combinations of processing steps, but it enables the creation of different fractions with different physicochemical properties useful for food applications.

Serum proteins are extracted along with monolayer cells in plasticware and interfere with protein analysis.

Washing and lysing monolayer cells directly from cell culture plasticware is a commonly used method for protein extraction. We found that multiple protein bands were enriched in samples with low cell numbers from the 6-well plate cultures. These proteins contributed to the overestimation of cell proteins and led to the uneven protein loading in Western blotting analysis. In Coomassie blue stained SDS-PAGE gels, the main enriched protein band is about 69 kDa and it makes up 13.6% of total protein from 104 U251n cells. Analyzed by mass spectrometry, we identified two of the enriched proteins: bovine serum albumin and bovine serum transferrin. We further observed that serum proteins could be extracted from other cell culture plates, dishes and flasks even after washing the cells 3 times with PBS. A total of 2.3 mg of protein was collected from a single well of the 6-well plate. A trace amount of the protein band was still visible after washing the cells 5 times with PBS. Thus, serum proteins should be considered if extracting proteins from plasticware, especially for samples with low cell numbers.

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles.

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16±7nm, and 19±4nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43±19nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.