Trypsin Affinity Chromatography Research Articles

Development of a rapid process for purification of Bowman-Birk and Kunitz inhibitors from legume seeds, and evaluation of their biophysical, insecticidal, and antimicrobial properties

Bowman-Birk inhibitor (BBI ~10 kDa) and Kunitz inhibitor (KI ~20 kDa) are serine protease/proteinase inhibitor(s) [PI(s)] ubiquitously found in several Leguminous plant species with insecticidal and therapeutic properties. Due to narrow molecular mass differences, the separation of these inhibitors from a single seed variety is tedious. The present study is aimed to develop a rapid protocol (<24 h) for purifying BBI and KI from legume seeds using mild trichloroacetic acid (TCA) extraction followed by trypsin-affinity chromatography. The mature seeds of Vigna radiata and Cajanus platycarpus are used as a model to purify BBI and KI using this protocol. The BBI and KI purified from the seeds of V. radiata are labeled as VrBBI & VrKI, and C. platycarpus are labeled as CpBBI & CpKI, respectively. These PIs are confirmed by immunodetection and MALDI-TOF studies and further characterized for their structural (CD & fluorescence spectroscopy) and functional properties (temperature & DTT stability). BBI(s) purified using the above process are effective in the management of castor semi-looper ‘Achaea janata’, while KI(s) are effective in the management of pod borer ‘Helicoverpa armigera’. Besides, both BBI(s) and KI(s) have significant potential in controlling the growth of methicillin-sensitive ‘Staphylococcus aureus’, a gram-positive pathogenic bacterium.

Proteinase inhibitors of pigeonpea cv. BSMR 736: Characterization and bioefficacy against Helicoverpa armigera

Pigeonpea is an agriculturally important leguminous crop with high vulnerability to insect pest attack specifically, Helicoverpa armigera. The proteinase inhibitors (PIs) mediated host plant resistance against insect pests is a promising sustainable agricultural research practice. The current study was carried out to perceive biochemical characterization of proteinase inhibitors named PPTI in the pigeonpea (cv. BSMR 736). The purification of PPTI from crude protein seed extract was achieved by acetone precipitation, N-LP-IEF, and trypsin affinity chromatography. It was found to inhibit bovine trypsin and HaGPs in vitro. The optimal conditions for inhibition were pH 8 and temperature 40ºC. The PPTI showed four isoinhibitors bands on native, non-reducing and reducing SDS-PAGE in the range of 26.7–19.3 KDa. Upon resolution on two-dimensional gel electrophoresis (2-DE), PPTI produced nine pI variant spots having isoelectric point (pI) 6.6, 6.6, 6.3, 6.1, 5.9, 5.8, 5.7, 5.6 and 5.6. An artificial diet containing PPTI reduced the H. armigera larval weight about 69%, with 25% mortality. For eco-friendly sustainable agricultural practices, natural compounds like PPTI could be expressed in transgenic crops to prevent the invasion of H. armigera in pigeonpea.

Anti-hyperglycemic Properties of a Purified Proteinaceous Protease Inhibitor from Macrotyloma Uniflorum Seeds

Fabaceae are a rich source of protease inhibitors. A proteinaceous protease inhibitor of 25 KDa designated as Macrotyloma uniflorum protease inhibitor (MUPI) was isolated from seeds of Macrotyloma uniflorum and purified to homogeneity by ammonium sulphate precipitation, DEAE Sepharose column and CNBr activated Sepharose 4B Trypsin affinity chromatography. The purity was checked by reverse phase HPLC. Long-term type 2 diabetes can lead to various biological complications, such as hypertension and heart-related diseases. The glucose uptake studies were carried out with the purified MUPI and it reveals its potential to be explored as a potent anti-hyperglycemic agent. MUPI was rendered safer on HepG2 cells after MTT cytotoxicity assay. The results of glucose uptake studies suggest MUPI to be equally efficient to that of the positive control drug Metformin against diabetes mellitus. The significance of plant-based inhibitors for modulating carbohydrate breakdown and control of glycemic index is explored to reduce the risk factors and side effects of the available drugs.

Antifungal and Antimicrobial Properties of a Purified Protease Inhibitor from Macrotyloma Uniflorum Seeds.

Plant protease Inhibitors (PIs) play key roles in regulation of many biological activities and being less toxic, more potent and specific in comparision to chemical inhibitors. A new proteinaceous trypsin inhibitor was isolated and purified from Macrotyloma uniflorum seeds with a molecular mass of 25 KDa was purified to homogeneity via three sequential purification steps i.e., ammonium sulphate precipitation to CNBr activativated Sepharose 4B coupled trypsin affinity chromatography. Purified protease inhibitor (PI) showed optical specific activities of 665 µmols of tyrosine released/ml/min. Overall, there was a remarkable increase in the fold of purification. MUTI is stable to denaturation by heat (upto 80°C), pH (4-10).The inhibitory activity increased to two fold in the presence of mercuric chloride and got reduced by half in the presence of ferric chloride. SDS, Dithiothreitol, β-mercaptoethanol, Hydrogen peroxide, Triton X 100 enhanced its inhibitory activity whereas activity was reduced in the presence of DMSO. Chemical modification of the inhibitor by DEPC decreased its activity but the activity was increased considerably when modified with NE and PMSF. Presence of protease inhibitor activity was confirmed by reverse zymography and Dot- blot. Also MUPI has antifungal and antimicrobial properties. MUPI inhibited Phytophthora capsici by 16.6% and Rhizoctonia solani by 27.7%. Antibacterial activity was shown against Staphylococcus aureus and Pseudomonas aeruginosa. MUPI retained 90% inhibition upon storage at 4°C for over a period of six months. Thus PI can be effectively exploited to increase the shelf life of seafood.

Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata

Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest – Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ± 11 nM) and chymotrypsin (Ki of 446 ± 9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50 = 8 μg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.

Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions

We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K(i) in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family.

Purification of three major forms of β-hCG from urine and production of polyclonal antibodies against them

Objectives This study has been conducted to develop a proper and relatively simple method for purification of three major forms of β-hCG from urine to use as clinical calibrators and also to produce anti-β-hCG polyclonal antibodies against these purified forms. Design and methods Major β-hCG forms from urine of pregnant women were purified using an ultrafiltration method followed by three successive steps: 1) affinity chromatography on a Concanavalin A-Sepharose column, 2) ion exchange chromatography on a DEAE-Sephacel column and 3) preparative gel electrophoresis. Specific polyclonal antibodies against the purified forms were produced by immunizing rats. We also extracted β-hCG from urine using trypsin affinity chromatography. Results Three β-hCG forms with the molecular mass (MW) of 28, 32 and 35 kDa were purified and identified by ELISA method using monoclonal antibodies against two distinct epitopes (β2 and β11) on β-hCG. The titer of prepared antibodies was comparable with common standard anti-β-hCG antibodies. Also we could purify a 37 kDa form of β-hCG by trypsin affinity chromatography. Conclusions Here we proposed two different proper methods for purification of major forms of β-hCG from urine. We also found that three prepared β-hCG forms expose the β11 epitope and have an hCGβcf epitope expressed. Specific antibodies against purified β-hCG forms had comparable titers and could be used in diagnostic kits.

A trypsin and chymotrypsin inhibitor from Caesalpinia bonduc seeds: Isolation, partial characterization and insecticidal properties

Evolution of proteinase inhibitor diversity in leguminous plants of tropical rainforests is under immense pressure from the regular upregulation of proteolytic machinery of their pests. The present study illustrates the isolation and bioinsecticidal potency of a serine proteinase inhibitor from the seeds of Caesalpinia bonduc (CbTI), inhabiting Great Nicobar Island, India. Following initial fractionation by ammonium sulfate precipitation, CbTI was purified to homogeneity by ion exchange, gel filtration and trypsin affinity chromatography. SDS–PAGE of gel filtrated CbTI showed a couple of proteins CbTI-1 (∼16 kDa) and CbTI-2 (20 kDa) under non-reducing conditions, which subsequent to trypsin affinity chromatography yielded only CbTI-2. Both Native PAGE as well as iso-electric focusing showed 2 iso-inhibitors of CbTI-2 (p I values of 5.35 and 4.6). CbTI exhibited tolerance to extremes of temperatures (0–60 °C) and pH (1–12). A 1:1 stoichiometric ratio was noted during CbTI-2–trypsin complex formation, which was absent on binding with chymotrypsin. Further, SDS–PAGE analysis also showed that CbTI-1 has affinity only towards chymotrypsin, whereas both trypsin and chymotrypsin formed complexes with CbTI-2. Dixon plot analysis of CbTI-2 yielded inhibition constants ( K i) of 2.75 × 10 −10 M and 0.95 × 10 −10 M against trypsin and chymotrypsin activity respectively. Preliminary investigations on the toxicological nature of CbTI revealed it to be a promising bioinsecticidal candidate.

Purification of a Novel, Heat-Stable Serine Protease Inhibitor Protein from Ovaries of the Desert Locust,Schistocerca gregaria

A protease-inhibitor was isolated from mature ovaries ofSchistocerca gregariaby a combination of trypsin-affinity chromatography and reverse-phase high performance liquid chromatography. It was characterized by aminoterminal amino acid sequencing using Edman degradation based automated microsequencing and by MALDI-TOF mass spectrometry. The N-terminal sequence (Y)XAEXDELA(A)EEY(Y)Q(Q)X(I)(L)M (X being a Cys, an irregular or modified amino acid) revealed no similarities with any other protease inhibitors isolated from invertebrate or vertebrate source. The 14 kDa inhibitor was found to be heat-stable. It shows potent inhibitory activity toward bovine trypsin and chymotrypsin, but not toward pancreatic elastase. It is likely that the characterized inhibitor will serve as an important tool for understanding its role in insect development.

A mutant trypsin-like enzyme from Streptomyces fradiae, created by site-directed mutagenesis, improves affinity chromatography for protein trypsin inhibitors.

The Ser-170 residue of a trypsin-like enzyme from Streptomyces fradiae (SFT), which is considered to be the active-site serine, was replaced with alanine by site-directed mutagenesis to improve the affinity chromatography step for a Kazal-type trypsin inhibitor pancreatic secretory trypsin inhibitor (PSTI). The resulting mutant SFT, designated as [S170A]SFT, was expressed in Streptomyces lividans and purified to homogeneity. [S170A]SFT was catalytically inactive, but still had the ability to bind tightly to PSTI and to soybean trypsin inhibitor with dissociation constants of 3.1 x 10(-7) M and 1.9 x 10(-8) M respectively. We further demonstrated that recombinant human PSTI secreted into Saccharomyces cerevisiae culture broth could be purified to homogeneity with a one-step [S170A]SFT-affinity column. The purified PSTI contained no molecules intramolecularly cleaved by active trypsin, which are found when trypsin-affinity chromatography is used for the purification. This eliminated the need for further separation of intact PSTI from intramolecularly cleaved PSTI by high-performance liquid chromatography, thus simplifying and improving its purification process.

Affinity chromatography of trypsin using chitosan as ligand support

Chitosan beads were prepared for use as affinity adsorbent carrier. The affinity ligand, chicken ovomucoid, was immobilized on the chitosan via a cross-linker, glutaraldehyde. The results showed that 60 mg chicken ovomucoid could be immobilized on 1 g chitosan, and the maximum binding capacity for trypsin was about 8·104 U/g dry adsorbent. The procedure for preparing the chitosan-based affinity adsorbents was much safer and simpler than when a Sepharose-based matrix was the support. Columns packed with the affinity adsorbents were employed for trypsin chromatography. The experimental results revealed that the affinity adsorbents possessed good mechanical strength and storage stability and could be also operated repeatedly. Chitosan was suitable for use as an affinity adsorbent support for laboratory-scale and large-scale purification.

Primary structure of trypsin inhibitors from Sicyos australis

Three trypsin inhibitors from Sicyos australis, have been isolated, purified and sequenced. Following protein extraction with ammonium sulphate, the mixture of inhibitors was separated from other proteins by trypsinaffinity chromatography. Subsequent purification of the individual inhibitors was accomplished by reversed-phase HPLC. The primary structures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensive sequence similarity with inhibitors from cultivated Cucurbitaceae species, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the use of MS-MS was crucial to the elucidation of its primary structure. ESI-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.

Purification and identification of a novel and four known serine proteinase inhibitors secreted by human glioblastoma cells.

Our previous studies have shown that some human cancer cell lines produce pancreatic trypsinogen, plasminogen, and tissue-type kallikrein. To understand the regulatory mechanism of these proteinases, serine proteinase inhibitors secreted by human glioblastoma cell line T98G were analyzed by gelatin reverse zymography with trypsin. The serum-free conditioned medium of T98G cells showed more than ten trypsin inhibitor bands ranging from 16 to 150 kDa in the reverse zymography. Major trypsin inhibitors were purified by trypsin-affinity chromatography. Analysis of their N-terminal amino acid sequences demonstrated that the purified inhibitors were identical to the secreted forms of amyloid protein precursors (APPs), tissue factor pathway inhibitor (TFPI), placental protein 5 (PP5)/TFPI-2, and secretory leukocyte proteinase inhibitor (SLPI). In addition, a novel 25-kDa trypsin-binding protein, tentatively named p25TI, was identified. p25TI showed weak inhibitory activity against trypsin in reverse zymography as compared with the other inhibitors. The secretion of multiple forms of serine proteinase inhibitors by human cancer cells raises the possibility that they might be involved in the abnormal growth of cancer cells.

Potent and Selective Kunitz Domain Inhibitors of Plasma Kallikrein Designed by Phage Display

Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.

Proteinase inhibitor II is developmentally regulated in Nicotiana flowers

Proteinase inhibitors (PI) are expressed in several members of the Solanaceae. According to species, PI can be both developmentally and environmentally regulated. We investigated the presence and developmental regulation of PI in the flowers of Nicotiana plumbaginifolia. By trypsin affinity chromatography a small protein, Np‐PI‐II, was isolated. This protein had an N‐terminus similar to a part of a Nicotiana alata PI‐II, and an inhibitory activity towards trypsin. Inhibitory activity is expressed at very early stages of floral development. No inhibitor is present in the vegetative meristems. The inhibitory activity remains high throughout flower maturation and rapidly declines in the ovary after pollination. In the stigma of mature Nicotiana tabacum flowers trypsin inhibitory activity is detected extracellularly. Proteinase inhibitor activity is not a regular feature of the stigmas of several solanaceous genera.Proteinase inhibitors apparently have no function in the development of floral structures and probably act as protective agents against fungal infection of and insect damage to the floral parts and the developing fruit.

Kunitz domain inhibitors of tissue factor-factor VIIa. I. Potent inhibitors selected from libraries by phage display.

Potent active-site inhibitors of human tissue factor-Factor VIIa (TF.FVIIa) have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries displayed on phage. Eight randomized positions on the extended primary binding loop (P5 through P4') and positions 34 and 39 were examined in three separate libraries. Libraries contained from 3.2 x 10(5) to 3.2 x 10(6) potential variants resulting from replacing up to 5 positions with all 20 amino acids. Following 4 rounds of selection against FVIIa associated with immobilized tissue factor (TF), 12 clones from each library were sequenced. Variants were purified by trypsin affinity chromatography and reverse-phase high performance liquid chromatography, and characterized for their ability to inhibit TF.FVIIa chromogenic activity. Measured apparent equilibrium dissociation constants (Ki*) ranged from about 10 to 500 nM. From sequence and activity data, an overall consensus sequence, TF7I-C, was constructed by site-directed mutagenesis. TF7I-C differed from APPI at 4 key residues, T11P, M17L, S19L, and G39Y, and inhibited TF.FVIIa with a Ki* = 1.9 +/- 0.4 nM, which represented an increase in binding affinity of more than 150-fold compared to APPI. At 40 microM, TF7I-C prolonged the clotting times 3.5-fold in a prothrombin time assay and > 10-fold at 7 microM in an activated partial thromboplastin time assay. Prolongation of the activated partial thromboplastin time correlates with potent inhibition of FXIa (Ki* = 0.8 nM) and plasma kallikrein (Ki* = 1.2 nM). TF7I-C also inhibited plasmin (Ki* = 40 nM) and FXa (Ki* = 55 nM), but not activated protein C, thrombin, or FXIIa (Ki* > 10 microM each).

A new protein inhibitor of trypsin and activated hageman factor from pumpkin (Cucurbita maxima) seeds

A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 × 10−8 and 4.1 × 10−8 M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.

Neutral proteinases of the human intervertebral disc

Disc tissue consisting of pooled annuli fibrosus and nuclei pulposus from the cadaver of an adolescent aged 19 years was extracted with 4.0 M Gu-HCl. Proteins of low buoyant density ( ϱ ≤ 1.38 g/ml) containing the disc enzymes and inhibitors were separated from proteoglycans of high buoyant density ( ϱ ≥ 1.50 g/ml) by density gradient ultracentrifugation. Sephadex G-75F gel chromatography followed by trypsin affinity chromatography was then used to resolve disc proteolytic and trypsin inhibitory activities. The results obtained were strongly suggestive of the presence of a high molecular weight zymogen which upon activation generated a population of smaller molecular weight proteinases. The disc proteinases obtained by this process showed similar properties in terms of: (a) their pH optima (7.4–7.6); (b) their inhibition patterns by class-specific proteinase inhibitors; (c) their variation of activity as a function of NaCl and lysine concentrations; and (d) the hydrodynamic size of their proteoglycan degradation products. The activated disc neutral proteinase demonstrated many characteristics in common with plasmin; however, unlike the latter, the disc proteinases also showed some calcium dependence.

Affinity chromatography of trypsin on casein precipitated with trichloroacetic acid

Affinity chromatography of trypsin on casein precipitated with trichloroacetic acid

Affinity chromatography of trypsin on thermally modified casein

Animal, plant and microbial proteases can be isolated and purified using a variety of methods. Affinity chromatography is of special interest because it allows the desired protease to be isolated in a single step. In this method various substrates, substrate analogues and inhibitors (both natural and synthetic) or antibodies are used as ligands for immobilization on various types of insoluble supports1-3. In contrast, an insoluble protein substrate such as collagen can be used for the affinity chromatography of collagenase itself without immobilization on to an insoluble matrix4. This is a simplification of the chromatographic method because the coupling procedure may be a critical step. To simplify the affinity chromatography of trypsin, a method was developed using thermally modified casein as a water-insoluble protease substrate. The method is inexpensive because only granulated casein is used to prepare an affinity chromatographic sorbent.