Crystal structures of human serum albumin in complex with lysophosphatidylcholine.

Lysophospholipids (lysoPLs) are crucial metabolites involved in various physiological and pathological cellular processes. Understanding their binding interactions, particularly with human serum albumin (HSA), is essential due to their role in regulating lysoPLs-induced cytotoxicity. However, the precise mechanism of lysoPLs binding to HSA remains elusive. In this study, we employed fluorescence quenching and optical interferometry assays to demonstrate direct binding between lysophosphatidylcholine (LPC) and HSA (KD = 25 µM). Furthermore, we determined crystal structures of HSA in complex with LPC, both in the absence and the presence of the endogenous fatty acid myristate (14:0). The crystal structure of binary HSA:LPC revealed that six LPC molecules are bound to HSA at the primary fatty acid binding sites. Interestingly, the ternary HSA:Myr:LPC structure demonstrated the continued binding of three LPC molecules to HSA at binding sites 1, 3, and 5 in the presence of myristate. These findings support HSA's role as a carrier protein for lysoPLs in blood plasma and provide valuable insights into the structural basis of their binding mechanisms.

The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism.

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.

Nafamostat Mesylate in Combination with the Mouse Amino-Terminal Fragment of Urokinase-Human Serum Albumin Improves the Treatment Outcome of Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is highly aggressive and causes a higher proportion of metastatic cases. However, therapies directed to specific molecular targets have rarely achieved clinically meaningful improvements in the outcome of TNBC therapy. A urokinase-type plasminogen activator (uPA), one of the best-validated biomarkers of breast cancer, is an extracellular proteolytic serine protease involved in many pathological and physiological processes, including tumor cell invasion and metastasis. Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases and has been used as a therapeutic agent for the treatment of TNBC. Nevertheless, NM has poor specificity for serine proteases and is easy be hydrolyzed; moreover, the inhibitory mechanism of TNBC therapy is unclear. In this study, we combine NM with a macromolecular drug delivery vehicle, mouse amino-terminal fragment of urokinase-human serum albumin (mATF-HSA), to form a complex (mATF-HSA:NM) using the dilution-incubation-purification method. mATF specifically targets uPAR overexpressed on the surface of TNBC cells; moreover, HSA prevents NM from being hydrolyzed by numerous serine proteases. mATF-HSA:NM showed stronger inhibitory effects on the proliferation and metastasis of TNBC in vitro and in vivo without significant cytotoxicity on normal cells and tissues. In addition, we demonstrated that NM mediates metastasis of TNBC cells through inhibition of uPA using a stable uPA knockdown cell line (MDA-MB231 shuPA). Overall, we have developed a macromolecular complex targeted to treat high uPAR-expressing tumor types, and mATF-HSA can potentially be used to load other types of drugs with tumor-targeting specificity for mouse tumor models and is a promising tool to study tumor biology in mouse tumor models.

Potential Roles and Future Perspectives of Chitinase 3-like 1 in Macrophage Polarization and the Development of Diseases.

Chitinase-3-like protein 1 (CHI3L1), a chitinase-like protein family member, is a secreted glycoprotein that mediates macrophage polarization, inflammation, apoptosis, angiogenesis, and carcinogenesis. Abnormal CHI3L1 expression has been associated with multiple metabolic and neurological disorders, including diabetes, atherosclerosis, and Alzheimer's disease. Aberrant CHI3L1 expression is also reportedly associated with tumor migration and metastasis, as well as contributions to immune escape, playing important roles in tumor progression. However, the physiological and pathophysiological roles of CHI3L1 in the development of metabolic and neurodegenerative diseases and cancer remain unclear. Understanding the polarization relationship between CHI3L1 and macrophages is crucial for disease progression. Recent research has uncovered the complex mechanisms of CHI3L1 in different diseases, highlighting its close association with macrophage functional polarization. In this article, we review recent findings regarding the various disease types and summarize the relationship between macrophages and CHI3L1. Furthermore, this article also provides a brief overview of the various mechanisms and inhibitors employed to inhibit CHI3L1 and disrupt its interaction with receptors. These endeavors highlight the pivotal roles of CHI3L1 and suggest therapeutic approaches targeting CHI3L1 in the development of metabolic diseases, neurodegenerative diseases, and cancers.

Monomer and Oligomer Transition of Zinc Phthalocyanine Is Key for Photobleaching in Photodynamic Therapy.

Photodynamic therapy (PDT) is recognized as a powerful method to inactivate cells. However, the photosensitizer (PS), a key component of PDT, has suffered from undesired photobleaching. Photobleaching reduces reactive oxygen species (ROS) yields, leading to the compromise of and even the loss of the photodynamic effect of the PS. Therefore, much effort has been devoted to minimizing photobleaching in order to ensure that there is no loss of photodynamic efficacy. Here, we report that a type of PS aggregate showed neither photobleaching nor photodynamic action. Upon direct contact with bacteria, the PS aggregate was found to fall apart into PS monomers and thus possessed photodynamic inactivation against bacteria. Interestingly, the disassembly of the bound PS aggregate in the presence of bacteria was intensified by illumination, generating more PS monomers and leading to an enhanced antibacterial photodynamic effect. This demonstrated that on a bacterial surface, the PS aggregate photo-inactivated bacteria via PS monomer during irradiation, where the photodynamic efficiency was retained without photobleaching. Further mechanistic studies showed that PS monomers disrupted bacterial membranes and affected the expression of genes related to cell wall synthesis, bacterial membrane integrity, and oxidative stress. The results obtained here are applicable to other types of PSs in PDT.

Structural study of the uPA-nafamostat complex reveals a covalent inhibitory mechanism of nafamostat.

Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases produced during the coagulation cascade and inflammation. Previous studies showed that NM was a highly safe drug for the treatment of different cancers, but the precise functions and mechanisms of NM are not clear. In this study, we determined a series of crystal structures of NM and its hydrolysates in complex with a serine protease (urokinase-type plasminogen activator [uPA]). These structures reveal that NM was cleaved by uPA and that a hydrolyzed product (4-guanidinobenzoic acid [GBA]) remained covalently linked to Ser195 of uPA, and the other hydrolyzed product (6-amidino-2-naphthol [6A2N]) released from uPA. Strikingly, in the inactive uPA (uPA-S195A):NM structure, the 6A2N side of intact NM binds to the specific pocket of uPA. Molecular dynamics simulations and end-point binding free-energy calculations show that the conf1 of NM (6A2N as P1 group) in the uPA-S195A:NM complex may be more stable than conf2 of NM (GBA as P1 group). Moreover, in the structure of uPA:NM complex, the imidazole group of His57 flips further away from Ser195 and disrupts the stable canonical catalytic triad conformation. These results not only reveal the inhibitory mechanism of NM as an efficient serine protease inhibitor but also might provide the structural basis for the further development of serine protease inhibitors.

Structural Basis of Covalent Inhibitory Mechanism of TMPRSS2-Related Serine Proteases by Camostat.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.

A general strategy to inhibit serine protease by targeting its autolysis loop.

Serine proteases are a large family of enzymes critical for multiple physiological processes, and proven diagnostic and therapeutic targets in several clinical indications. The high similarity of active sites among different serine proteases posts a challenge to reach high selectivity for inhibitors of serine proteases targeting at the active site. Here, we demonstrated that one particular surface loop on serine proteases (autolysis loop) can be used to regulate their catalytic activity, through surveying the recent works including ours, and such an approach can reach high specificity. The autolysis loop is highly variable among different serine proteases, explaining the high specificity of inhibitors targeting the autolysis loop. We also outline the structural origin that links the perturbation of the autolysis loop and the inhibition of protease activity. Thus, the autolysis loop appears to be a highly sensitive allosteric site and can be used as a general handle to develop pharmacological agents to intervene with the activities of serine proteases in, eg, blood coagulation.

Role of Angiopoietin-Tie axis in vascular and lymphatic systems and therapeutic interventions.

The Angiopoietin (Ang)-Tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie) axis is an endothelial cell-specific ligand-receptor signaling pathway necessary for vascular and lymphatic development. The Ang-Tie axis is involved in regulating angiogenesis, vascular remodeling, vascular permeability, and inflammation to maintain vascular quiescence. Disruptions in the Ang-Tie axis are involved in many vascular and lymphatic diseases and play an important role in physiological and pathological vascular processes. Given recent advances in the Ang-Tie axis in the vascular and lymphatic systems, this review focuses on the multiple functions of the Ang-Tie axis in inflammation-induced vascular permeability, vascular remodeling, atherosclerosis, ocular angiogenesis, tumor angiogenesis, and metastasis. A summary of relevant therapeutic approaches to the Ang-Tie axis, including therapeutic antibodies, recombinant proteins and small molecule drugs are also discussed. The purpose of this review is to provide new hypotheses and identify potential therapeutic strategies based on the Ang-Tie signaling axis for the treatment of vascular and lymphatic-related diseases.

A new class of quaternary ammonium compounds as potent and environmental friendly disinfectants.

Quaternary ammonium compounds (QACs) are inexpensive and readily available disinfectants, and have been widely used, especially since the COVID-19 outbreak. The toxicity of QACs to humans has raised increasing concerns in recent years. Here, a new type of QACs was synthesized by replacing the alkyl chain with zinc phthalocyanine (ZnPc), which consists of a large aromatic ring and is hydrophobic in nature, similar to the alkyl chain of QACs. Three ZnPc-containing disinfectants were synthesized and fully characterized. These compounds showed 15-16 fold higher antimicrobial effect against Gram-negative bacteria than the well-known QACs with half-maximal inhibitory (IC50) values of 1.43 µM, 2.70 µM, and 1.31 µM, respectively. With the assistance of 680 nm light, compounds 4 and 6 had much higher bactericidal toxicities at nanomolar concentrations. Compound 6 had a bactericidal efficacy of close to 6 logs (99.9999% kill rate) at 1 µM to Gram-positive bacteria, including MRSA, under light illumination. Besides, these compounds were safe for mammalian cells. In a mouse model, compound 6 was effective in healing wound infection. Importantly, compound 6 was easily degraded at working concentrations under sunlight illumination, and is environmentally friendly. Thus, compound 6 is a novel and promising disinfectant.

Regulation of pro-σK activation: a key checkpoint in Bacillus subtilis sporulation.

The Gram-positive bacterium Bacillus subtilis initiates the sporulation process under conditions of nutrient limitation. Here, we review related work in this field, focusing on the protein processing of the pro-σK activation. The purpose of this review is to illustrate the mechanism of pro-σK activation and provide structural insights into the regulation of spore production. Sporulation is not only important in basic science but also provides mechanistic insight for bacterial control in applications in, e.g., food industry.

Structural comparison of CD163 SRCR5 from different species sheds some light on its involvement in porcine reproductive and respiratory syndrome virus-2 infection in vitro.

Porcine reproductive and respiratory syndrome (PRRS) is a serious disease burdening global swine industry. Infection by its etiological agent, PRRS virus (PRRSV), shows a highly restricted tropism of host cells and has been demonstrated to be mediated by an essential scavenger receptor (SR) CD163. CD163 fifth SR cysteine-rich domain (SRCR5) is further proven to play a crucial role during viral infection. Despite intense research, the involvement of CD163 SRCR5 in PRRSV infection remains to be elucidated. In the current study, we prepared recombinant monkey CD163 (moCD163) SRCR5 and human CD163-like homolog (hCD163L1) SRCR8, and determined their crystal structures. After comparison with the previously reported crystal structure of porcine CD163 (pCD163) SRCR5, these structures showed almost identical structural folds but significantly different surface electrostatic potentials. Based on these differences, we carried out mutational research to identify that the charged residue at position 534 in association with the one at position 561 were important for PRRSV-2 infection in vitro. Altogether the current work sheds some light on CD163-mediated PRRSV-2 infection and deepens our understanding of the viral pathogenesis, which will provide clues for prevention and control of PRRS.

Potent inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 by photosensitizers compounds.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) posed a major challenge to the public health. Currently, no proven antiviral treatment for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is available. Here we report compounds pentalysine ß-carbonylphthalocyanine zinc (ZnPc5K) and chlorin e6 (ce6) potently inhibited the viral infection and replication in vitro with EC50 values at nanomolar level. These compounds were first identified by screening a panel of photosensitizers for photodynamic viral inactivation. Such viral inactivation strategy is implementable, and has unique advantages, including resistance to virus mutations, affordability compared to the monoclonal antibodies, and lack of long-term toxicity.

Specifically targeting cancer proliferation and metastasis processes: the development of matriptase inhibitors.

Matriptase is a type II transmembrane serine protease, which has been suggested to play critical roles in numerous pathways of biological developments. Matriptase is the activator of several oncogenic proteins, including urokinase-type plasminogen activator (uPA), hepatocyte growth factor (HGF) and protease-activated receptor 2 (PAR-2). The activations of these matriptase substrates subsequently lead to the generation of plasmin, matrix metalloproteases (MMPs), and the triggers for many other signaling pathways related to cancer proliferation and metastasis. Accordingly, matriptase is considered an emerging target for the treatments of cancer. Thus far, inhibitors of matriptase have been developed as potential anti-cancer agents, which include small-molecule inhibitors, peptide-based inhibitors, and monoclonal antibodies. This review covers established literature to summarize the chemical and biochemical aspects, especially the inhibitory mechanisms and structure-activity relationships (SARs) of matriptase inhibitors with the goal of proposing the strategies for their future developments in anti-cancer therapy.

Insight to the residue in P2 position prevents the peptide inhibitor from being hydrolyzed by serine proteases.

Peptidic inhibitors of proteases are attracting increasing interest not only as drug candidates but also for studying the function and regulation mechanisms of these enzymes. Previously, we screened out a cyclic peptide inhibitor of human uPA [Formula: see text] and found that Ala substitution of P2 residue turns upain-1 to a substrate. To further investigate the effect of P2 residue on the peptide behavior transformation, we constructed upain-1-W3F, which has Phe replacement in the P2 position. We determined KD and Ki of upain-1-W3F and found that upain-1-W3F might still exist as an inhibitor. Furthermore, the high-resolution crystal structure of upain-1-W3F·uPA reveals that upain-1-W3F indeed stays as an intact inhibitor bind to uPA. We thus propose that the P2 residue plays a nonnegligible role in the conversion of upain-1 to a substrate. These results also proposed a strategy to optimize the pharmacological properties of peptide-based drug candidates by hydrophobicity and steric hindrance.Abbreviations : uPA: urokinase-type plasminogen activator; SPD: serine protease domain; S1 pocket: specific substrate-binding pocket.

Structural determination of group A Streptococcal surface dehydrogenase and characterization of its interaction with urokinase-type plasminogen activator receptor.

Streptococcus pyogenes (group A Streptococcus, GAS) has caused a wide variety of human diseases. Its multifunctional surface dehydrogenase (SDH) is crucial for GAS life cycle. Furthermore, GAS infection into human pharyngeal cells has been previously shown to be mediated by the interaction between SDH and host urokinase-type plasminogen activator receptor (uPAR). However, the structural information of SDH remains to be elucidated and there are few detailed studies to characterize its interaction with uPAR. In-depth research on these issues will provide potential targets and strategies for combating GAS. Here, we prepared recombinant SDH tetramer in Escherichia coli BL21 (DE3) cells. After purification and crystallization, we determined its crystal structure at 1.74 Å. The unique characteristics might be potentially explored as drug targets or vaccine immunogen. We subsequently performed gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and in vitro pull-down analyses. The results showed that their interaction was too weak to form stable complexes and the role of uPAR involved in GAS infection needs further demonstration. Altogether the current work provides the first view of SDH and deepens the knowledge of GAS infection.

6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease.

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Ki = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1ß subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.

The crystal structure of a multidomain protease inhibitor (HAI-1) reveals the mechanism of its auto-inhibition.

Hepatocyte growth factor activator inhibitor 1 (HAI-1) is a membrane-bound multidomain protein essential to the integrity of the basement membrane during placental development and is also important in maintaining postnatal homeostasis in many tissues. HAI-1 is a Kunitz-type serine protease inhibitor, and soluble fragments of HAI-1 with variable lengths have been identified in vivo The full-length extracellular portion of HAI-1 (sHAI-1) shows weaker inhibitory activity toward target proteases than the smaller fragments, suggesting auto-inhibition of HAI-1. However, this possible regulatory mechanism has not yet been evaluated. Here, we solved the crystal structure of sHAI-1 and determined the solution structure by small-angle X-ray scattering. These structural analyses revealed that, despite the presence of long linkers, sHAI-1 exists in a compact conformation in which sHAI-1 active sites in Kunitz domain 1 are sterically blocked by neighboring structural elements. We also found that in the presence of target proteases, sHAI-1 adopts an extended conformation that disables the auto-inhibition effect. Our results also reveal the roles of non-inhibitory domains of this multidomain protein and explain the low activity of the full-length protein. The structural insights gained here improve our understanding of the regulation of HAI-1 inhibitory activities and point to new approaches for better controlling these activities.

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection.

Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus. IMPORTANCE: PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides structural information on the CD163 SRCR domain for the first time but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.

A novel purification procedure for recombinant human serum albumin expressed in Pichia pastoris.

Plasma-derived human serum albumin (pHSA) has important applications in many clinical indications, including blood loss, serious burn, or hemorrhagic shock. The limited supply and potential infectious pathogen contamination of pHSA have stimulated the development of recombinant human serum albumin (rHSA). However, rHSA often entraps endogenous or exogenous impurities, including color pigments and polysaccharides. Therefore, the purification of rHSA to high purity remains the bottleneck in large-scale production of rHSA. Herein we report a novel two-step purification protocol for rHSA generated from Pichia pastoris. In the first step, rHSA was partially unfolded to expose impurities entrapped into rHSA and was then removed. In the second step, rHSA was crystallized to further remove impurities. Through this procedure, the pigment content (A350/A280) and polysaccharides content of rHSA were reduced to 0.0141 and 0.253 µg/mg, respectively, which were comparable to pHSA. In addition, the secondary structure and bioactivity of purified rHSA are preserved. The purification procedure developed in this study is a simple, short and low cost method to produce active rHSA, or rHSA-fusion proteins, to high purity, and also suitable for the purification of other disulfide-rich proteins.