Expression, purification and structural characterization of resveratrol synthase from Polygonum cuspidatum.

Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit.

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Plant and algal lysophosphatidic acid acyltransferases increase docosahexaenoic acid accumulation at the sn-2 position of triacylglycerol in transgenic Arabidopsis seed oil.

Although docosahexaenoic acid (DHA), an important dietary omega-3 polyunsaturated fatty acid (PUFA), is at present primarily sourced from marine fish, bioengineered crops producing DHA may offer a more sustainable and cost-effective source. DHA has been produced in transgenic oilseed crops, however, DHA in seed oil primarily occupies the sn-1/3 positions of triacylglycerol (TAG) with relatively low amounts of DHA in the sn-2 position. To increase the amount of DHA in the sn-2 position of TAG and in seed oil, putative lysophosphatidic acid acyltransferases (LPAATs) were identified and characterized from the DHA-producing alga Schizochytrium sp. and from soybean (Glycine max). The affinity-purified proteins were confirmed to have LPAAT activity. Expression of the Schizochytrium or soybean LPAATs in DHA-producing Arabidopsis expressing the Schizochytrium PUFA synthase system significantly increased the total amount of DHA in seed oil. A novel sensitive band-selective heteronuclear single quantum coherence (HSQC) NMR method was developed to quantify DHA at the sn-2 position of glycerolipids. More than two-fold increases in sn-2 DHA were observed for Arabidopsis lines expressing Schizochytrium or soybean LPAATs, with one Schizochytrium LPAAT driving DHA accumulation in the sn-2 position to 61% of the total DHA. Furthermore, expression of a soybean LPAAT led to a redistribution of DHA-containing TAG species, with two new TAG species identified. Our results demonstrate that transgenic expression of Schizochytrium or soybean LPAATs can increase the proportion of DHA at the sn-2 position of TAG and the total amount of DHA in the seed oil of a DHA-accumulating oilseed plant. Additionally, the band-selective HSQC NMR method that we developed provides a sensitive and robust method for determining the regiochemistry of DHA in glycerolipids. These findings will benefit the advancement of sustainable sources of DHA via transgenic crops such as canola and soybean.

Functional Characterization of an Anthocyanin Dimalonyltransferase in Maize.

Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3-O-glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3-O-glucoside tested. Results of this study provide insight into the structure-function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases.

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8.

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.

The expression and purification of LpxA of Chlamydia trachomatis and preparation of its polyclonal antibody.

The purpose of this study is to purify the LpxA protein of Chlamydia trachomatis (Ct) and prepare the polyclonal antibody against LpxA protein, so as to lay a foundation for studying the function of LpxA protein. The LpxA gene was amplified by PCR. The expression plasmid pET28a-LpxA was constructed by using pET28a as the vector. The fusion protein containing 6 histidine tag was induced by IPTG and purified by Ni2+ chromatography gel. The purified His-LpxA protein was used as an immunogen to immunize New Zealand rabbits subcutaneously through the back to prepare polyclonal antibody. Immunoblotting was used to detect the reaction between the antibody and His-LpxA. The determination of polyclonal antibody titer was detected by ELISA. The relative molecular weight of His-LpxA was 32.8 kDa, and it could be expressed in Escherichia coli. The purity of the purified protein was about 95%. After immunizing New Zealand rabbits, the antiserum was able to recognize the recombinant His-LpxA protein with a titer greater than 1:10240. In this study, LpxA protein was successfully purified and antiserum was prepared, which provided an experimental basis for studying the function of LpxA protein.

Purification of Recombinant DHHC Proteins Using an Insect Cell Expression System.

DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5 mg of DHHC3 and 1.0 mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1 mg/mL by SEC.

Biochemical Assays for Ghrelin Acylation and Inhibition of Ghrelin O-Acyltransferase.

Ghrelin O-acyltransferase (GOAT) is an enzyme responsible for octanoylating and activating ghrelin, a peptide hormone that plays a key role in energy regulation and hunger signaling. Due to its nature as an integral membrane protein, GOAT has yet to be purified in active form which has complicated biochemical and structural studies of GOAT-catalyzed ghrelin acylation. In this chapter, we describe protocols for efficient expression and enrichment of GOAT in insect cell-derived microsomal fraction, HPLC-based assays for GOAT acylation activity employing fluorescently labeled peptides, and assessment of inhibitor potency against GOAT.

Identifying Lysophosphatidic Acid Acyltransferaseâ€ ß (LPAAT-ß) as the Target of a Nanomolar Angiogenesis Inhibitor from a Phenotypic Screen Using the Polypharmacology Browser PPB2.

By screening a focused library of kinase inhibitor analogues in a phenotypic co-culture assay for angiogenesis inhibition, we identified an aminotriazine that acts as a cytostatic nanomolar inhibitor. However, this aminotriazine was found to be completely inactive in a whole-kinome profiling assay. To decipher its mechanism of action, we used the online target prediction tool PPB2 (http://ppb2.gdb.tools), which suggested lysophosphatidic acid acyltransferaseâ€…ß (LPAAT-ß) as a possible target for this aminotriazine as well as several analogues identified by structure-activity relationship profiling. LPAAT-ß inhibition (IC50 ≈15 nm) was confirmed in a biochemical assay and by its effects on cell proliferation in comparison with a known LPAAT-ß inhibitor. These experiments illustrate the value of target-prediction tools to guide target identification for phenotypic screening hits and significantly expand the rather limited pharmacology of LPAAT-ß inhibitors.

Identification and heterologous reconstitution of a 5-alk(en)ylresorcinol synthase from endophytic fungus Shiraia sp. Slf14.

A new type III polyketide synthase gene (Ssars) was discovered from the genome of Shiraia sp. Slf14, an endophytic fungal strain from Huperzia serrata. The intron-free gene was cloned from the cDNA and ligated to two expression vectors pET28a and YEpADH2p-URA3 for expression in Escherichia coli BL21(DE3) and Saccharomyces cerevisiae BJ5464, respectively. SsARS was efficiently expressed in E. coli BL21(DE3), leading to the synthesis of a series of polyketide products. Six major products were isolated from the engineered E. coli and characterized as 1,3-dihydroxyphenyl-5-undecane, 1,3-dihydroxyphenyl-5-cis-6'-tridecene,1,3-dihydroxyphenyl-5-tridecane, 1,3-dihydroxyphenyl-5-cis-8'-pentadecene, 1,3-dihydroxyphenyl-5-pentadecane, and 1,3-dihydroxyphenyl-5-cis-10'-heptadecene, respectively, based on the spectral data and biosynthetic origin. Expression of SsARS in the yeast also led to the synthesis of the same polyketide products, indicating that this enzyme can be reconstituted in both heterologous hosts. Supplementation of soybean oil into the culture of E. coli BL21(DE3)/SsARS increased the production titers of 1-6 and led to the synthesis of an additional product, which was identified as 5-(8'Z,11'Z-heptadecadienyl) resorcinol. This work thus allowed the identification of SsARS as a 5-alk(en)ylresorcinol synthase with flexible substrate specificity toward endogenous and exogenous fatty acids. Desired resorcinol derivatives may be synthesized by supplying corresponding fatty acids into the culture medium.

Characterization of a Novel Thermostable Enzyme from Thermus sp. 2.9 with Phospholipase and Acyltransferase Activities.

Phospholipases are classified in different enzyme families according to the ester bond they cleave within phospholipids. The use of phospholipases in industrial processes has prompted the search for new enzymes with differential properties. A gene encoding a novel phospholipase (PLP_2.9) was identified in the genome of the thermophilic strain Thermus sp. 2.9. The analysis of the primary sequence unveiled a patatin-like domain. The alignment of the amino acid sequence of PLP_2.9 to other bacterial patatin-related proteins showed that the four blocks characteristic of this type of phospholipases and the amino acids representing the catalytic dyad are conserved in this protein. PLP_2.9 was overexpressed in Escherichia coli and the purified enzyme was characterized biochemically. PLP_2.9 hydrolyzed p-nitrophenyl palmitate at alkaline pH over a wide range of temperatures (55-80°C), showing high thermostability. PLP_2.9 displayed phospholipase A and acyltransferase activities on egg yolk phosphatidylcholine. Due to its high thermostability, PLP_2.9 has potential applications as a catalyst in several industrial processes.

Identification and Characterization of a Novel Palmitoyl Acyltransferase as a Druggable Rheostat of Dynamic Palmitoylome in L. donovani.

Palmitoylation has been recently identified as an important post-translational rheostat for controlling protein function in eukaryotes. However, the molecular machinery underlying palmitoylation remains unclear in the neglected tropical parasite, Leishmania donovani. Herein, we have identified a catalog of 20 novel palmitoyl acyltransferases (PATs) and characterized the promastigote-specific PAT (LdPAT4) containing the canonical Asp-His-His-Cys (DHHC) domain. Immunofluorescence analysis using in-house generated LdPAT4-specific antibody demonstrated distinct expression of LdPAT4 in the flagellar pocket of promastigotes. Using metabolic labeling-coupled click chemistry method, the functionality of this recombinant enzyme could be authenticated in E. coli strain expressing LdPAT4-DHHC domain. This was evident by the cellular uptake of palmitic acid analogs, which could be successfully inhibited by 2-BMP, a PAT-specific inhibitor. Using CSS-Palm based in-silico proteomic analysis, we could predict up to 23 palmitoylated sites per protein in the promastigotes, and further identify distinctive palmitoylated protein clusters involved in microtubule assembly, flagella motility and vesicular trafficking. To highlight, proteins such as Flagellar Member proteins (FLAM1, FLAM5), Intraflagellar Transport proteins (IFT88), and flagellar motor assembly proteins including the Dynein family were found to be enriched. Furthermore, analysis of global palmitoylation in promastigotes using Acyl-biotin exchange purification identified a set of S-palmitoylated proteins overlapping with the in-silico proteomics data. The attenuation of palmitoylation using 2-BMP demonstrated several phenotypic alterations in the promastigotes including distorted morphology, reduced motility (flagellar loss or slow flagellar beating), and inefficient invasion of promastigotes to host macrophages. These analyses confirm the essential role of palmitoylation in promastigotes. In summary, the findings suggest that LdPAT4 acts as a functional acyltransferase that can regulate palmitoylation of proteins involved in parasite motility and invasion, thus, can serve as a potential target for designing chemotherapeutics in Visceral Leishmaniasis.

Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens.

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene (phaCPf) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (phaCPf+, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (phaCPf+) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaCPf, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

Purification and characterization of 1-acyl-sn-glycerol-3-phosphate acyltransferase with a substrate preference for polyunsaturated fatty acyl donors from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10.

1-Acyl-sn-glycerol-3-phosphate acyltransferase (designated as PlsC in bacteria) catalyzes the acylation of lysophosphatidic acid and is responsible for the de novo production of phosphatidic acid, a precursor for the synthesis of various membrane glycerophospholipids. Because PlsC is an integral membrane protein, it is generally difficult to solubilize it without causing its inactivation, which has been hampering its biochemical characterization despite its ubiquitous presence and physiological importance. Most biochemical studies of PlsC have been carried out using crude membrane preparations or intact cells. In this study, we succeeded in solubilization and purification of a recombinant PlsC in its active form from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10 using 6-cyclohexyl-1-hexyl-ß-d-maltoside as the detergent. We characterized the purified enzyme and found that it has a substrate preference for the acyl donors with a polyunsaturated fatty acyl group, such as eicosapentaenoyl group. These results provide a new method for purification of the PlsC family enzyme and demonstrate the occurrence of a new PlsC with unique substrate specificity.

Isolation and functional characterization of hydroxycinnamoyltransferases from the liverworts Plagiochasma appendiculatum and Marchantia paleacea.

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT, EC: 2.3.1.133) is a key metabolic entry point for the synthesis of monolignols in vascular plants; however, little is known about HCT in liverworts. Here, the isolation and characterization of HCTs encoded by the two liverwort species, Plagiochasma appendiculatum and Marchantia paleacea, are described. The sequences of the two enzymes harbor features typical of BAHD family members, except for the presence of a stretch of >100 residues that are not represented in higher plant HCTs. When truncated versions of both genes, which were constructed to clarify the significance of these extra residues, were investigated, it became apparent that the full-length and the truncated gene products shared similar catalytic activity and recognized the same substrates in vitro. They also functioned equivalently in vivo either when transiently expressed in tobacco to cause a higher total production of CGA (5-CQA) and 4-CQA or stably expressed in liverworts to accumulate the lignin-like contents. A structural model of MpHCT suggests that its active site bind to its substrate similar to that of Arabidopsis thaliana HCT. While truncated forms of HCT were deposited in the nucleocytoplasm, the full-length versions occurred exclusively in the cytoplasm. The conclusion is that liverworts produce bona fide HCTs that represent a point of departure in studying the evolution of lignin synthesis in plants.

In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria.

Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1SG(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACSPt) and CT from T. thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60°C and 75°C, respectively. The in vitro PHA synthesis was successfully performed by ACSPt, CTTt, PhaC1SG(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45°C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37°C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures.

Identification of a new polyhydroxyalkanoate (PHA) producer Aquitalea sp. USM4 (JCM 19919) and characterization of its PHA synthase.

Aquitalea sp. USM4 (JCM 19919) was isolated from a freshwater sample at Lata Iskandar Waterfall in Perak, Malaysia. It is a rod-shaped, gram-negative bacterium with high sequence identity (99%) to Aquitalea magnusonii based on 16S rRNA gene analysis. Aquitalea sp. USM4 also possessed a PHA synthase gene (phaC), which had amino acid sequence identity of 77-78% to the PHA synthase of Chromobacterium violaceum ATCC12472 and Pseudogulbenkiania sp. NH8B. PHA biosynthesis results showed that wild-type Aquitalea sp. USM4 was able to accumulate up to 1.5 g/L of poly(3-hydroxybutyrate), [P(3HB)]. The heterologous expression of the PHA synthase gene of Aquitalea sp. USM4 (phaCAq) in Cupriavidus necator PHB-4 had resulted in PHA accumulation up to 3.2 g/L of P(3HB). It was further confirmed by 1H nuclear magnetic resonance (NMR) analysis that Aquitalea sp. USM4 and C. necator PHB-4 transformant were able to produce PHA containing 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB) and 3-hydroxy-4-methylvalerate (3H4MV) monomers from suitable precursor substrates. Interestingly, relatively high PHA synthase activity of 863 U/g and 1402 U/g were determined in wild-type Aquitalea sp. USM4 and C. necator PHB-4 transformant respectively. This is the first report on the member of genus Aquitalea as a new PHA producer as well as in vitro and in vivo characterization of a novel PHA synthase from Aquitalea sp. USM4.

Discovery of a new polyhydroxyalkanoate synthase from limestone soil through metagenomic approach.

PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated.

Combining a PagP fusion protein system with nickel ion-catalyzed cleavage to produce intrinsically disordered proteins in E. coli.

Many proteins contain intrinsically disordered regions that are highly solvent-exposed and susceptible to post-translational modifications. Studying these protein segments is critical to understanding their physiologic regulation, but proteolytic degradation can make them difficult to express and purify. We have designed a new protein expression vector that fuses the target protein to the N-terminus of the integral membrane protein, PagP. The two proteins are connected by a short linker containing the sequence SRHW, previously shown to be optimal for nickel ion-catalyzed cleavage. The methodology is demonstrated for an intrinsically disordered segment of cardiac troponin I. cTnI[135-209]-SRHW-PagP-His6 fusion protein was overexpressed in Escherichia coli, accumulating in insoluble inclusion bodies. The protein was solubilized, purified using nickel affinity chromatography, and then cleaved with 0.5mM NiSO4 at pH 9.0 and 45 °C, all in 6M guanidine-HCl. Nickel ion-catalyzed peptide bond hydrolysis is an effective chemical cleavage technique under denaturing conditions that preclude the use of proteases. Moreover, nickel-catalyzed cleavage is more specific than the most commonly used agent, cyanogen bromide, which cleaves C-terminal to methionine residues. We were able to produce 15 mg of purified cTnI[135-209] from 1L of M9 minimal media using this protocol. The methodology is more generally applicable to the production of intrinsically disordered protein segments.

Immobilization of active lipase B from Candida antarctica on the surface of polyhydroxyalkanoate inclusions.

Polyhydroxyalkanoate (PHA) beads, recombinantly produced in Escherichia coli, were functionalized to display lipase B from Candida antarctica as translational protein fusion. The respective beads were characterized in respect to protein content, functionality, long term storage capacity and re-usability. The direct fusion of the PHA synthase, PhaC, to lipase B yielded active PHA lipase beads capable of hydrolyzing glycerol tributyrate. Lipase B beads showed stable activity over several weeks and re-usability without loss of function.