Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae

A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter. The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein. A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor. From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA. The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step. The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.     

High level soluble production of functional ribonuclease inhibitor in Escherichia coli by fusing it to soluble partners

Ribonuclease inhibitor (RI) is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases which inhibits ribonucleolytic activity. Expression of recombinant RI is extremely difficult to reach high levels in soluble form in the cytoplasm of Escherichia coli. Here, we utilized five N-terminal fusion partners to improve the soluble expression of RI. Among these five fusion partners which have been screened, maltose-binding protein (MBP), N-utilization substance A (NusA) and translation initiation factor 2 domain I (IF2) have greatly improved the soluble expression level of recombinant murine RI under the drive of T7 promoter, while glutathione S-transferase (GST) and small ubiquitin modifying protein (SUMO) were much less efficient. All these RI-fusion proteins remained to be highly active in inhibiting RNase A activity. Furthermore, all fusion tags can be efficiently removed by enterokinase digestion to generate native RI which results the highest yield to date (>30mg of native RI per liter culture). And a convenient two-step immobilized metal affinity chromatography (IMAC) method has been implemented in our study, comparing with the traditional RNase A affinity chromatography method.     

High-level soluble expression of bioactive porcine myostatin propeptide in E. coli

Myostatin (MSTN) is a potent negative regulator of skeletal muscle mass. The activity of MSTN is suppressed by MSTN propeptide (MSTNPro), the N-terminal part of unprocessed MSTN that is cleaved off during posttranslational MSTN processing. Easy availability of MSTNPro would help to investigate the potential of the protein as an agent to enhance muscle growth in agricultural animal species. Thus, this study was designed to produce bioactive wild-type porcine MSTN propeptide (pMSTNProW) and its mutated form at the BMP-1/TLD proteolytic cleavage site (pMSTNProM) in Escherichia coli. The pMSTNProW and pMSTNProM genes were separately cloned into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene and were transformed and expressed in soluble forms in E. coli. For each milliliter of cell culture, about 40 μg of soluble MBP-pMSTNProW and MBP-pMSTNProM proteins were purified by amylose resin affinity chromatography. Further purification by anion exchange chromatography of the affinity-purified fractions yielded about 10 μg/mL culture of MBP-pMSTNProW and MBP-pMSTNProM proteins. Factor Xa protease cleaved the fusion partner MBP from MBP-pMSTNPro proteins, and approximately 4.2 μg of pMSTNProW and pMSTNProM proteins were purified per milliliter of culture. MBP-pMSTNProM was resistant to digestion by BMP-1 metalloproteinase, while MBP-pMSTNProW was cleaved into two fragments by BMP-1. Both MBP-pMSTNProW and MBP-pMSTNProM demonstrated their MSTN binding affinities in a pulldown assay. In an in vitro gene reporter assay, both proteins inhibited MSTN bioactivity without a significant difference in their inhibitory capacities, indicating that the cell culture-based gene reporter assay has limitation in detecting the true in vivo biological potencies of mutant forms of MSTNPro proteins at the BMP-1/TLD cleavage site. Current results show that a high-level production of bioactive porcine MSTNpro is possible in E. coli, and it remains to be investigated whether the administration of the MSTNpro can improve skeletal muscle growth in pigs via suppression of MSTN activity in vivo.     

Isolation and characterization of monoclonal antibodies against ribonuclease inhibitor

Mouse monoclonal antibodies were generated against human ribonuclease inhibitor, an intracellular regulatory protein. A total of four antibodies were isolated, all of which were of the immunoglobulin G1 subtype. Western blot analysis of the antibodies suggested monospecificity. Based on immunoradiometric competition assays two of the antibodies were determined to be directed against the same antigenic epitope, while the other two were against a second and possibly third epitope. None of the antibodies appeared to be directed against the ribonuclease binding site of the antigen. Data is presented suggesting that ribonuclease inhibitor is present in normal human serum. The potential significance of these findings is discussed.     

Material characterization of porcine lenticular soluble proteins

The soluble proteins present in the ocular lens impart important optical and dynamic mechanical properties on the lens. The short-range order of crystallin proteins grants transparency to a very concentrated protein solution. This unique protein system directly enables proper visual function of the eye. These proteins were investigated in steady and oscillatory shear. Steady shear data were fitted with a modified Herschel-Bulkley yield stress model that allows for a Newtonian plateau at low shear rates. The Cox-Merz rule was used in conjunction with large amplitude oscillatory shear to give insight into the degradation of the fluid structure with increasing strain. The shear thinning viscoelastic behavior of these proteins gives rise to beneficial mechanical properties and results from the same short-range order granting optical transparency.     

Isolation and partial characterization of ribonuclease inhibitor from goat liver

Ribonuclease inhibitor (RI), a 50 kDa protein, has been found both in mammalian and nonmammalian tissues. We have isolated RI from goat liver and partial characterization has been accomplished. For the isolation of RI, DEAE cellulose column chromatography followed by affinity chromatography using CNBr activated Sepharose 4B was performed. The inhibition of ribonucleolytic activity of Ribonuclease A has been checked by an agarose gel based assay. The antiangiogenic property of the protein was tested by the chorioallantoic membrane (CAM) assay. Results indicate inhibition of angiogenesis.     

Cloning and characterization of a ribonuclease L inhibitor from the silkworm, Bombyx mori.

The ribonuclease L (RNase L) pathway plays an important role in the response of cells to double-stranded RNA (dsRNA) during the events such as virus infection. Ribonuclease L inhibitor (RLI) belonging to the ABC transporter family is known as a regulator of the RNase L pathway. The homologs of RLI were reported in many organisms including the fruit fly and mosquito, but their functions in insects and arthropods have not been elucidated to date. In the present study, we cloned a cDNA of a silkworm RLI homolog, termed BmRLI, and its nucleotide sequence was determined. RT-PCR analysis revealed that the expression of BmRLI mRNA was marked in the testis, ovary and fat body. From the cDNA, recombinant protein with an apparent molecular mass of 69 kDa was expressed in Escherichia coli and cultured insect cells. Although no obvious effect of up-regulation of the BmRLI expression on RNAi was observed, its down-regulation slightly reduced RNAi efficiency.     

High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris

Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition. Pichia pastoris was used as a host to efficiently express the pST gene in this study. Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures. SDS-PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of approximately 25 kDa, and had the same immunoreactivity as native pST. The culture supernatant of our rpST expression strain, X-33/pPICZalphaA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography. MALDI-TOF-MS demonstrated a molecular mass of 21,771Da for rpST, close to its predicted size. Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a pI value between 4.55 and 5.2. Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection. These results indicate that the P. pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels.     

Purification and characterization of a carbonic anhydrase II inhibitor from porcine plasma.

Plasma from many vertebrates, including pigs, contains a soluble component that inhibits the CO2 hydrase activity of carbonic anhydrase (CA). This activity was purified to homogeneity (approximately 4000-fold) from porcine plasma using a combination of DEAE-Affi-Gel Blue chromatography and carbonic anhydrase II-affinity chromatography, yielding 16 mg of inhibitory protein/L of plasma. This protein, porcine inhibitor of carbonic anhydrase (pICA), is a monomeric protein with an apparent molecular mass of 79 kDa, as determined by electrospray mass spectrometry. As isolated, pICA contains about 3 kDa of N-linked glycosylation removable by peptide N-glycosidase F. pICA inhibits CA reversibly with a 1:1 stoichiometry. pICA is a potent and specific inhibitor of the CA II isozyme, with Ki < 0.1 nM for porcine CA II at pH 7.4. Although the Ki is dependent on the CA isozyme type (CA II < CA IV < CA III approximately CA I), it is relatively insensitive to the species source, as long as it is mammalian. The Ki is pH dependent with log Ki decreasing linearly as the pH decreases, implicating at least one ionizable group with the pKa < or = 6.5 in the binding interaction. The isozyme and species dependence of the inhibition suggest that pICA interacts with amino acids on the surface of CA II.     

High-level production of soluble adenine nucleotide translocator from Schistosoma japonicum in E. coli cell-free system

Adenine nucleotide translocator is an important member of the mitochondrial carrier family, which is involved in the transportation of various metabolites. In the present work, the Escherichia coli cell-free system was chosen as an alternative way to express this highly toxic membrane protein. The expression level of insoluble sjANT from Schistosoma japonicum in E. coli cell-free system could attain 472 μg/mL, about 25.2 times improvement over the previous report. The obtained insoluble sjANT can be resolubilized with different detergents. Among them, Digitonin could effectively solubilize approximate 38% of the target membrane protein. Moreover, sjANT can be further expressed in the hydrophobic E. coli cell-free system as a soluble form with presence of different detergents. The results suggested that Digitonin and Brij 58 were two ideal candidates to support high expression of soluble sjANT, and the highest soluble expression level (182 μg/mL) was achieved with the supplementation of 0.4% (v/v) Digitonin in the cell-free system. The present work has provided a rapid and efficient procedure to express the complex and highly toxic membrane protein in the cell-free system, and will be beneficial to construct a novel drug-discovery model to screen the sjANT-based inhibitors for schistosomiasis treatment.     

Folding mechanism of porcine ribonuclease

The kinetics of unfolding and refolding of porcine ribonuclease were investigated. The unfolded state of this protein was found to consist of a fast-refolding species (UF) and two slow-refolding species (UIS and UIIS). After the rapid collapse of the structure during the N (native)----UF unfolding reaction, UIS and UIIS are produced from UF by two independent slow isomerizations of the unfolded polypeptide chain, leading ultimately to a mixture of about 10% UF, 20% UIIS and 70% UIS molecules at equilibrium. This is at variance with all other ribonucleases investigated to date, which show a distribution of 20% UF, 60 to 70% UIIS and only 10 to 20% UIS. The two isomerizations of the unfolded porcine protein differ strongly in rate. The first process with tau = 250 seconds (10 degrees C) leads to an increase in the fluorescence of Tyr92 and was identified as cis in equilibrium trans isomerization of Pro93. At equilibrium, most unfolded molecules contain an incorrect trans Pro93. The second isomerization is much slower (tau = 1300 s at 10 degrees C) and leads to a predominance of the incorrect isomer as well. Like isomerization of Pro93, it is governed by an activation enthalpy of 22 kcal/mol (92 kJ/mol) and it was tentatively assigned to the Pro114-Pro115 sequence of porcine ribonuclease. Because of the wide separation in rate between the two reactions, molecules with an incorrect isomer only at Pro93 accumulate transiently after unfolding. These are the UIIS molecules. Most of them are finally converted to UIS by the 1300 second process. All molecules that have undergone this isomerization refold very slowly, i.e. the UIS molecules. The major fraction contains two incorrect isomers. A 1300 second isomerization after unfolding and a predominant very slow refolding reaction were observed only for the porcine protein. We suggest that these changes in the folding mechanism may be correlated with the presence of the Pro114-Pro115 sequence, which occurs only in porcine ribonuclease. The refolding pathway of porcine UIIS involves the rapid formation of a native-like intermediate with an incorrect trans Pro93 as was found previously for the bovine ribonuclease, where the UIIS species predominates in the unfolded state.     

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.     

Inactivation of ribonuclease inhibitor by thiol-disulfide exchange.

Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.     

Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression

Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37°C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34°C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl β-d-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.     

Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor

The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the complex in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 degrees C, this value is 1.3 X 10(-7) s-1 (t1/2 congruent to 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 X 10(-16) M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 X 10(-5) s-1 (t1/2 = 13 h), and the Ki value is 4.4 X 10(-14) M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2'-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2'-phosphate decrease the association rate in a manner consistent with a competitive mode of inhibition.     

Interaction of human placental ribonuclease with placental ribonuclease inhibitor

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.     

Purification and characterization of alkaline ribonuclease inhibitor from normal and nephrotic rat kidney compared to inhibitor from rat liver

Alkaline ribonuclease inhibitor has been purified from the kidney of control rats as well as from the kidney of nephrotic rats and the properties of the kidney inhibitor have been compared to those of the ribonuclease inhibitor obtained from the liver of normal control rats. For kidney inhibitor, the molecular weight (50,000), the electrophoretic mobility, the sensitivity to heat, to sulfhydryl reactants, and to tryptic digestion are all the same as those of liver inhibitor. The specific activity and properties of the purified inhibitor obtained from nephrotic kidney were the same as for inhibitor from control kidney, but the yield of inhibitor protein was three times greater from nephrotic kidney. Evidence for the view that there is an increased synthesis of ribonuclease inhibitor in nephrotic kidney is discussed.