Lisa Wen

Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.

Nucleotide sequence of a rice genomic clone that encodes a class I endochitinase

TTGCTGAAGATCGATGCACCATGCA TATCCATCTCTATATAAAGCCATGCGATCCCACCGATTCTTGCACACACACTAGCTACTT CTACTTCTATCATACCAAACAAACTAGCTTAATTTGCATTGCATCACATTGCCGGCCGCC ATGAGAGCTCTCGCTCTCGCGGTGGTGGCCATGGCGGTGGTGGCCGTGCGCGGCGAGCAG M R A L A L A V V A M A V V A V R G E Q TGCGGCAGCCAGGCCGGCGGCGCGCTCTGCCCCAACTGCCTCTGCTGCAGCCAGTACGGC C G S Q A G G A L C P N C L C C S Q Y G TGGTGCGGCTCCACCTCCGATTACTGCGGCGCCGGCTGCCAGAGCCAGTGCTCCGGCGGC W C G S T S D Y C G A G C Q S Q C S G G TGCGGCGGCGGCCCGACCCCGCCCTCCAGCGGTGGCGGCAGCGGCGTCGCCTCCATCATA C G G G P T P P S S G G G S G V A S I I TCGCCCTCGCTCTTCGACCAGATGCTGCTCCACCGCAACGACCAGGCGTGCCGCGCTAAG S P S L F D Q M L L H R N D Q A C R A K GGCTTCTACACCTACGACGCCTTCGTCGCCGCCGCCAACGCCTACCCGGACTTCGCCACC G F Y T Y D A F V A A A N A Y P D F A T ACGCGCGACGCCGACACCTGCAAGCGCGAGGTCGCCGCCTTCCTGGCGCAGACGTCCCAC T R D A D T C K R E V A A F L A Q T S H GAGACCACCGGCGGCTGGCCCACGGCGCCCGACGGCCCCTACTCCTGGGGCTACTGCTTC E T T G G W P T A P D G P Y S W G Y C F AAGGAGGAGAACAACGGCAACGCCCCCACATACTGCGAGCCCAAGCCGGAGTGGCCGTGC K E E N N G N A P T Y C E P K P E W P C GCCGCCGCGAAGAAGTACTACGGCCGGGGACCCATCCAGATCACCTACAACTACAACTAC A A A K K Y Y G R G P I Q I T Y N Y N Y GGCCGCGGGGCAGGCATCGGCTCCGACCTGCTCAACAACCCGGACCTGGTGGCGTCGGAC G R G A G I G S D L L N N P D L V A S D GCAGTCTCCTTCAAGACGGCGTTCTGGTTCTGGATGACGCCGCAGTCGCCCAAGCCGTCG A V S F K T A F W F W M T P Q S P K P S TGCCACGCGGTGATCACCGGCCAGTGGACGCCGTCCGCCGACGACCAGGCGGCGGGGCGC C H A V I T G Q W T P S A D D Q A A G R GTTCCGGGCTACGGCGAGATCACCAACATCATCAACGGCGGTGTGGAGTGCGGGCACGGC V P G Y G E I T N I I N G G V E C G H G GCGGACGACAAGGTGGCCGACCGGATCGGGTTCTACAAGCGCTACTGCGACATGCTGGGC A D D K V A D R I G F Y K R Y C D M L G GTCAGCTATGGCGATAACCTGGATTGCTACAACCAGAGGCCCTACCCGCCTTCCTAGTTG V S Y G D N L D C Y N Q R P Y P P S < ATATTTGATCCGAGCAGACGAATAAAATACAATGCACACGAGATTGTGAGACTCGTGAAA AACATATACTACCTCTGAATTTTAATACATATCTCTAAAACAAAGATTTGATCCGTTGAC CTGCAGGTCGAC 1237 Fig. 1.

A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice

A λgt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called α-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band inEco RI,Hind III, andBam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 microM with IC(50) value of 2 microM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 microM with IC(50) value of 6 microM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.

Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from Taiwanofungus camphorata.

Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli . Functional TcGrx was expressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed bands of approximately 15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (K(m)) values for beta-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 degrees C was 8.5 min, and its thermal inactivation rate constant K(d) was 6.52 x 10(-2) min(-1). The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.

Heterogeneity in cDNA clones encoding rice glutelin

Clones encoding the rice storage protein glutelin were selected from a cDNA library of immature rice seeds. Sequence analysis and hybridization studies on these clones provide insight into the nature of heterogeneity in glutelin genes. Based particularly on major differences in the 3′‐noncoding regions, it appears that glutelin genes fall into two sub‐families.

Ionic interactions between proteins in nonequilibrium pH gradient electrophoresis: Histones affect the migration of high mobility group nonhistone chromatin proteins

In two-dimensional gel electrophoresis of the high mobility group (HMG) proteins, it has proved necessary to use nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension rather than isoelectric focusing, because of the basic character of most of the HMG proteins [D. Tyrell, P. J. Isackson, and G. R. Reeck (1982) Anal. Biochem. 119, 433-439]. In this paper it is reported that in samples that contain histones, the mobilities of HMG proteins (particularly HMG-1, HMG-2, and HMG-E) are severely distorted in NEPHGE. This presumably results from formation of complexes between histones and HMG proteins through ionic interactions. Analysis of HMG proteins by NEPHGE/sodium dodecyl sulfate-gel electrophoresis is thus precluded in samples containing histones. Our results raise the possibility of similar artifacts occurring in NEPHGE (or isoelectric focusing) analysis of other proteins with regions of high charge density.

Monothiol Glutaredoxin cDNA from Taiwanofungus camphorata: A Novel CGFS-type Glutaredoxin Possessing Glutathione Reductase Activity

Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported monothiol Grxs. Two 3-D homology structures of the TcmonoGrx based on known structures of human Grx3 (pdb: 2DIY_A) and Mus musculus Grx3 (pdb: 1WIK_A) have been created. To characterize the TcmonoGrx protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C41(DE3). The recombinant His6-tagged TcmonoGrx was overexpressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed a predominant band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited glutathione reductase (GR) activity via dithionitrobenzoate (DTNB) assay. The Michaelis constant (K(M)) values for GSSG and NADPH were 0.064 and 0.041 mM, respectively. The enzymes half-life of deactivation at 60 °C was 10.5 min, and its thermal inactivation rate constant (k(d)) was 5.37 × 10(-2) min(-1). The enzyme was active under a broad pH range from 6 to 8. The enzyme retained 50% activity after trypsin digestion at 37 °C for 40 min. Both mutants C(40)→S(40) and C(165)→S(165) lost 40-50% GR activity, whereas the mutant S(168)→C(168) showed a 20% increase in its GR activity.

Modulation of Nitrosative Stress via Glutathione-Dependent Formaldehyde Dehydrogenase and S-Nitrosoglutathione Reductase

Glutathione-dependent formaldehyde dehydrogenase (GFD) from Taiwanofungus camphorata plays important roles in formaldehyde detoxification and antioxidation. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) against nitrosative stress. We investigated the modulation of HEK (human embryonic kidney) 293T cells under nitrosative stress by transfecting a codon optimized GFD cDNA from Taiwanofungus camphorata (Tc-GFD-O) to these cells. The parental and transfected HEK 293T cells were then subjected to S-nitrosoglutathione treatment to induce nitrosative stress. The results showed that in Tc-GFD-O-transfected 293T cells, the expression and activity of GFD increased. Additionally, these cells under the nitrosative stress induced by S-nitrosoglutathione showed both higher viability and less apoptosis than the parental 293T cells. This finding suggests that the Tc-GFD-O in HEK 293T cells may provide a protective function under nitrosative stress.

Molecular Cloning of Bovine eIF5A and Deoxyhypusine Synthase cDNA

Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (Nϵ-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we describe the cloning and characterization of bovine eIF5A and bovine deoxyhypusine synthase. The deduced bovine eIF5A protein is 100% identical to human eIF5A-1, and the deduced bovine deoxyhypusine synthase protein showed a 93% identity to the human protein.