Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe

The purification of large synthetic peptides using conventional separation techniques often results in poor yields and homogeneity due to the accumulation of chromatographically similar deletion and truncated impurities. We have developed a highly effective synthetic strategy and one-step purification procedure that is based on (i) the application of single coupling using HBTU/HOBt activation to reduce incomplete couplings, (ii) the use of N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping agent to terminate deletion sequences and (iii) the N-terminal derivatization of the complete peptidyl-resin with a reversible Fmoc-based chromatographic probe possessing enhanced physico-chemical properties (i.e. hydrophobicity, charge or affinity label). We report the application of a biotinylated probe, activated as the succinimidyl carbonate, for the purification of a 101 residue chaperonin protein from Rattus norvegicus (rat cpn10), previously synthesized using an optimized synthetic protocol. Biotinylated rat cpn10 was separated from underivatized impurities on an immobilized monomeric avidin column. Free rat cpn10 was released from avidin–agarose column with 5% aqueous triethylamine and after desalting by RP-HPLC gave 9.9% recovery. Characterization and assessment of homogeneity was achieved using ESI-MS, CZE and RP-HPLC.     

Site-specific biotinylation of human myeloid differentiation protein 88 in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Schneider 2 cell cytoplasm

In this work we describe the production of site-specific biotinylated human myeloid differentiation factor 88 (MyD88). A vector containing a coding sequence for a peptide derived from the carboxyl terminus of the Klebsiella pneumoniae oxalacetate decarboxylase α subunit was used to allow expression and biotinylation of MyD88 in Drosophila melanogaster Schneider 2 cell cytoplasm. As estimated by a comparison of Schneider 2 lysate with standard protein, the maximum expression level was 1.3 μg 10⁷ cells⁻¹. About 4 mg of biotinylated protein was purified by affinity chromatography on monomeric avidin from a I-L culture. Exogenous biotin added to the culture medium increased the biotinylation efficiency of the expressed protein. Biotinylated MyD88 produced in Drosophila cells was able to precipitate recombinant MyD88 expressed in human embryonic kidney cells. The stable expression of MyD88 in Drosophila Schneider 2 cells offers a convenient and attractive method for large-scale production, which may be required to clarify the role of MyD88 in the inflammatory response. Moreover, site-specific biotinylation of MyD88 provides a useful tag for interaction assays where high sensitivity is required.     

Oligopeptidase B from <Emphasis Type="Italic">Serratia proteamaculans</Emphasis>. I. Determination of primary structure,isolation, and purification of wild-type and recombinant enzyme variants

A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1–10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His₆-PSP was 1.5 mg/g biomass.     

Design,production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.     

Affinity purification of lipid vesicles

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.     

Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis

The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated. Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified. The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco). Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity. In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function. All mutants were also severely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains.To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli strain CG712 resulting in an inability to assemble -phage was exploited in a complementation assay. Transformation with plasmids directing the expression of mutant chloroplast cpn10 proteins in two cases restored -phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein. In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn 10 showed significantly reduced complementation efficiencies.     

Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

In this report, we describe a novel strategy for the design of a clinical urea biosensor using a process based on assembled multilayer systems. Biotinylated enzyme (urease–subiotin) was immobilized on the biotinylated polypyrrole coated Chemical field effect capacitance (ChemFEC) devices using the high avidin–biotin affinity. The immobilized enzyme activity was checked by its catalytic conversion of urea into carbon dioxide and ammonia. Electrochemical response of the bridge system constructed on Si/SiO₂/Si₃N₄ electrodes to urea addition was evaluated using the capacity–potential measurements. In addition, contact-angle measurements were carried out to control the change of surface energy and their components before and after each layer formation. The developed urea biosensor demonstrates high performances: a good sensitivity of 50 mV/pUrea in the linear urea concentration range from 10⁻⁴ to 10⁻¹ M and an excellent operational stability after three weeks of continuous use. The immobilized urease was characterised through its apparent Michaelis–Menten constant (5 mM) and the activation energy of the enzymatic reaction (20 kJ mol⁻¹). It was also shown that capacitive measurements can be used to quantify the interaction between molecular systems, based on avidin and biotin molecules.     

Intracellular production of a soluble and functional monomeric streptavidin in Escherichia coli and its application for affinity purification of biotinylated proteins

Monomeric forms of avidin and streptavidin [(strept)avidin] have many potential applications. However, generation of monomeric (strept)avidin in sufficient quantity is a major limiting factor. We report the successful intracellular production of an improved version of monomeric streptavidin (M4) in a soluble and functional state at a level of approximately 70 mg/L of an Escherichia coli shake flask culture. It could be affinity purified in one step using biotin agarose with 70% recovery. BIAcore biosensor analysis using biotinylated bovine serum albumin confirmed its desirable kinetic properties. Two biotinylated proteins with different degrees of biotinylation (5.5 and 1 biotin per protein) pre-mixed with cellular extracts from Bacillus subtilis were used to examine the use of M4-agarose in affinity purification of protein. Both biotinylated proteins could be purified in high purity with 75-80% recovery. With the mild elution and matrix regeneration conditions, the M4-agarose had been reused four times without any detectable loss of binding capability. The relatively high-level overproduction and easy purification of M4, excellent kinetic properties with biotinylated proteins and mild procedure for protein purification make vital advancements in cost-effective preparation of monomeric streptavidin affinity matrix with desirable properties for purification of biotinylated molecules.     

Localization of a multifunctional chaperonin (GroEL protein) in nitrogen-fixing Anabaena PCC 7120

The occurrence and distribution of a multifunctional chaperonin-60 (cpn60), the GroEL protein, was demonstrated in the cyanobacterium Anabaena PCC 7120 by using a rabbit anti-GroEL (Escherichia coli) antibody. Western-blot analysis showed a distinct cross-reaction with a protein of approx. 65 kilodaltons, analogous to the Mr of the E. coli homologue. Immunocyto-chemical studies of vegetative cells showed that a chaperonin was localized in both vegetative cells and heterocysts. In vegetative cells cpn60 was primarily detected both in the carboxysomes, and in the cytoplasm, though mainly in the thylakoid region of the latter. In heterocysts, specialized cells for nitrogen fixation, the cpn60 label was prominent and was evenly distributed throughout the cell. These results support recent findings that chaperonins are multifunctional proteins, and extend those findings by demonstrating the occurrence of cpn60 in a prokaryotic cyanobacterium and by raising the possibility of the involvement of this chaperonin in the assembly of heterocystous proteins.Abbreviations cpn60 chaperonin-60 - Mr relative molecular mass - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Protein H — a surface protein of Streptococcus pyogenes with separate binding sites for lgG and albumin

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (lgGFc) region of immunoglobulin (lg) G. In absorption experiments with human plasma, protein H–sepharose could absorb not only lgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 × 10⁹M⁻¹, which is higher than the affinity between lgG and protein H (K_a= 1.6 × 10⁹ M⁻¹). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein–protein interaction assays, the binding of albumin was mapped to three repeats (C1–C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and lgGFc-binding bacterial surface protein. Aiso lgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized lgG or lgGFc. This sequence was not found in previously described lgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify lgGFc- and albumin binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.     

An in vitro method for selective detection of free monomeric ubiquitin by using a C-terminally biotinylated form of ubiquitin

In an effort to design a selective assay allowing detection of free monomeric ubiquitin, an approach based on a C-terminally biotinylated form of ubiquitin is proposed. In the form of a polyubiquitin chain, ubiquitin marks proteins for degradation by the 26S proteasome. This covalently attached signal is assembled from multiple ubiquitins linked to each other via the C-terminus of one ubiquitin and the epsilon-amine of Lys48 of another ubiquitin. In the present study, a form of ubiquitin having the C-terminus modified with the addition of a biotinylation peptide tag was prepared. After expression, this modified ubiquitin was biotinylated in vitro using recombinant biotin ligase. Biotinylated ubiquitin was further purified using affinity chromatography on immobilized monovalent avidin. This tagged form of ubiquitin is blocked at the C-terminus and therefore can only act as an acceptor (Lys-48 donor) in polyubiquitin chain synthesis. In vitro enzymatic assembly of multiubiquitin chains from biotinylated monoubiquitin and natural monoubiquitin is demonstrated by Western blot analysis using horseradish peroxidase-conjugated streptavidin. Data obtained with this assay indicate potential uses of the C-terminally biotinylated form of ubiquitin for selective detection of monoubiquitin contamination in a cell extract experimentally depleted of ubiquitin, i.e. lysate Fraction II. Cell-free systems established for in vitro examination of ubiquitin involvement in proteolytic processes usually employ Fraction II, which should be essentially ubiquitin-free. It is suggested that the assay using biotinylated monoubiquitin can be useful to exclude the possibility that ubiquitin contamination of laboratory prepared lysate Fraction II accounts for protein degradation in this fraction.     

Single-chain antibody fragments specific to the hepatitis B surface antigen, produced in recombinant tobacco cell cultures

An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-histidine N-terminal tag was produced in cultured transgenic tobacco cells. Western blot and antigen-specific chromatography showed high levels of biologically active scFv in the culture supernatant (1 mg l^–1) and in cells (5 mg kg^–1). A simple one-step scFv purification was developed using immobilized metal ion affinity chromatography.     

Solid-phase biotinylation of antibodies

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.     

Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques

Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-Å resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.     

CD36 mediates binding of soluble thrombospondin-1 but not cell adhesion and haptotaxis on immobilized thrombospondin-1

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [¹²⁵I]-TSP1 and [¹²⁵I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats,^18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd.     

Purification and characterization of chaperonins 60 and 10 from Methylobacillus glycogenes

Two proteins belonging to the group I chaperonin family were isolated from an obligate methanotroph, Methylobacillus glycogenes. The two proteins, one a GroEL homologue (cpn60: M. glycogenes 60 kDa chaperonin) and the other a GroES homologue (cpn10: M. glycogenes 10 kDa chaperonin), composed a heteropolymeric complex in the presence of ATP. Both proteins were purified from crude extracts of M. glycogenes by anion-exchange (DEAE-Toyopearl) and gel-filtration (Sephacryl S-400) chromatography. The native molecular weights of each chaperonin protein as determined by high-performance liquid chromatography (HPLC) gel-filtration were 820 000 for cpn60 and 65 000 for cpnl0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the subunit molecular weights of cpn60 and cpnl0 were 58 000 and 10 000, respectively. Both cpn60 and cpnl0 possessed amino acid sequences which were highly homologous to other group I chaperonins. M. glycogenes cpn60 displayed an ATPase activity which was inhibited in the presence of cpn10. The chaperonins also displayed an ability to interact with and facilitate the refolding of Thermus malate dehydrogenase and yeast enolase in a manner similar to that of GroEL/ES. The similarities between the Escherichia coli GroE proteins are discussed.     

Two of the three <Emphasis Type="Italic">groEL</Emphasis> homologues in <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> are dispensable for normal growth

Although many bacteria contain only a single groE operon encoding the essential chaperones GroES and GroEL, examples of bacteria containing more than one groE operon are common. The root-nodulating bacterium Rhizobium leguminosarum contains at least three operons encoding homologues to Escherichia coli GroEL, referred to as Cpn60.1, Cpn60.2 and Cpn60.3, respectively. We report here a detailed analysis of the requirement for and relative levels of these three proteins. Cpn60.1 is present at higher levels than Cpn60.2, and Cpn60.3 protein could not be detected under any conditions although the cpn60.3 gene is transcribed under anaerobic conditions. Insertion mutations could not be constructed in cpn60.1 unless a complementing copy was present, showing that this gene is essential for growth under the conditions used here. Both cpn60.2 and cpn60.3 could be inactivated with no loss of viability, and a double cpn60.2 cpn60.3 mutant was also constructed which was fully viable. Thus only Cpn60.1 is required for growth of this organism.Dedicated to the memory of Professor V. Javier Benedí, 1957–2002     

A biotin-protein bond with stability in plasma

A nonradioactive label for peptide hormones would be useful for pharmacokinetic studies in infants, children, and pregnant women. Because the binding affinity between biotin and avidin is large (Ka=10(15) M(-1)), biotin could also serve as a covalent label for subsequent detection using a variety of avidin conjugates. However, biotin labels produced by most commercially available biotinylating reagents are rapidly cleaved from protein in plasma. We sought to synthesize a stable biotin label for protein. With the use of immunoglobulin G (IgG) as a model protein, biotin was conjugated through a cysteine residue; a carboxylate group was positioned alpha to the biotinamide bond. Stability of the bond in the presence of plasma and buffer control was assessed by release of biotin. Released biotin was separated from biotinylated IgG by ultrafiltration and was quantitated by an avidin-binding assay. In plasma, less than 0.6% of bound biotin was released. This release rate is not significantly different from buffer and is less than 7% of the release rate for IgG biotinylated by N-hydroxysuccinimide-LC-biotin. We conclude that this biotin-protein bond is stable in plasma. We speculate that many uses of avidin-biotin technology could be improved by using this method for protein labeling.     

Analysis of binding of biotinylated protoplast-release-inducing protein that induces release of gametic protoplasts in the Closterium peracerosum-strigosum-littorale complex

A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt⁺) cells and induces the release of gametic protoplasts from matingtype minus (mt⁻) cells of Closterium was biotinylated and then used to examine the interaction of this protein with mt⁻ cells. The protoplast-release-inducing activity of PR-IP was not altered after the biotinylation. When mt⁻ cells that had been pre-cultured for 24 h were incubated with biotinylated PR-IP for 6 h in nitrogen-deficient medium that contained 1% (w/v) bovine serum albumin, and then washed with the same medium, only a 19-kDa polypeptide, the smaller subunit of PR-IP, was detected in cells by the avidin and biotinylated horseradish-peroxidase macromolecular complex system. The amount of bound 19-kDa polypeptide increased with increasing doses of PR-IP and reached a maximum at around 10 nM, reflecting the protoplast-release-inducing activity. From a Scatchard plot, the dissociation constant of the polypeptide was calculated to be 2.7 · 10⁻⁸ M. The binding of the polypeptide proceeded only after an appropriate period of pre-culture in the light, and the polypeptide was competitively displaced by non-biotinylated PR-IP. From these results, it appears that the PR-IP induces the release of protoplasts from mt⁻ cells by binding of a polypeptide of relative molecular mass 19000 to the receptor on the cell surface in a manner analogous to the binding of peptide hormones in animals.     

In vivo biotinylation of recombinant beta-glucosidase enables simultaneous purification and immobilization on streptavidin coated magnetic particles

Beta-glucosidase from Bacillus licheniformis was in vivo biotinylated in Escherichia coli and subsequently immobilized directly from cell lysate on streptavidin coated magnetic particles. In vivo biotinylation was mediated by fusing the Biotin Acceptor Peptide to the C-terminal of beta-glucosidase and co-expressing the BirA biotin ligase. The approach enabled simultaneous purification and immobilization of the enzyme from crude cell lysate on magnetic particles because of the high affinity and strong interaction between biotin and streptavidin. After immobilization of the biotinylated beta-glucosidase the specific activity (using p-nitrophenyl-β-d-glucopyranoside as substrate) was increased 6.5 fold (compared to cell lysate). Immobilization of the enzyme resulted in improved thermal stability compared to free enzyme; after 2 h of incubation (at 50 °C) the residual enzyme activity of immobilized and free beta-glucosidase was 67 and 13%, respectively. The recyclability of immobilized beta-glucosidase was examined and it was observed that the enzyme could be recycled at least 9 times and retain 89% of its initial activity.