Molecular cloning and sequence analysis of human placental alkaline phosphatase

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.     

Bacterial expression of rat liver succinyl-CoA synthetase alpha-subunit. Factors that contribute to blocked translation of transcripts encoding a mitochondrial signal sequence

This study comprises a detailed evaluation of factors that are necessary to achieve high levels of expression of eukaryotic proteins in bacterial systems. We attempted to express a rat liver cDNA clone encoding the precursor to the alpha-subunit of succinyl-CoA synthetase in an Escherichia coli expression system, without success. Removal of the region encoding the mitochondrial signal peptide (115 nucleotides) allowed efficient expression of the mature protein. This nucleotide sequence was shown to block expression at the level of translation. Two regions within this fragment were able to block the expression of other genes such as E. coli lacZ. Inhibition of expression was due to the close proximity of these inhibitory sequences with the translation initiation region (TIR). Insertion of a spacer between the inhibitory sequence and the TIR relieved the block in translation. Analysis of the 115-nucleotide fragment identified sequences capable of extensive base-pairing with the Shine-Dalgarno and surrounding sequences. Such secondary structures are capable of blocking the formation of competent translation initiation complexes.     

Only the Mature Form of the Plastidic Chorismate Synthase Is Enzymatically Active

Coding regions of a cDNA for precursor and mature chorismate synthase (CS), a plastidic enzyme, from Corydalis sempervirens were expressed in Escherichia coli as translational fusions to glutathione-S-transferase. Fusion proteins were purified, and precursor and mature forms of CS were then released by proteolytic cleavage with factor Xa. Although mature CS was enzymatically active after release, activity could be detected neither for the precursor CS nor for corresponding glutathione-S-transferase fusion proteins. In contrast, two other shikimate pathway enzymes (shikimate kinase and 5-enol-pyruvylshikimate-3-phosphate synthase) have previously been shown to be as enzymatically active as their respective higher molecular weight precursors. By expression of unfused, mature CS from C. sempervirens in E. coli, it was possible to obtain large quantities of enzymatically active CS protein compared to yields from plant cell cultures. Expression levels in E. coli approached 1% of total soluble protein. No differences were found between authentic CS isolated from cell cultures and CS expressed in and purified from E. coli, which made possible a more detailed biochemical characterization of CS. Quaternary structure analysis of the purified mature CS indicated that the enzyme exists as a dimer, in contrast to the active tetrameric structures determined for E. coli and Neurospora crassa enzymes.     

Expression of cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase in Escherichia coli. Differences in kinetic mechanisms

The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase (E3) were expressed in Escherichia coli using a lambda PL promoter-driven prokaryotic expression vector. The expressed proteins in total cell extracts were identified by Western blot analysis using anti-pig heart E3 antibody and also by measurement of E3 activity. Most of the expressed human E3 polypeptides (five bands) were found in the insoluble pellet while primarily full-length mature E3 was found in the soluble fraction. About 2% of the total soluble protein was mature human E3 when expressed in wild type E. coli AR120. Since wild type E. coli has its own endogenous E3 activity, the expression of human E3 was performed in a pyruvate dehydrogenase complex-deficient strain of E. coli, JRG1342. The expressed recombinant human E3s in JRG1342 were purified to near homogeneity. The amino-terminal amino acid sequence analysis revealed that the recombinant mature E3 had an expected sequence while the recombinant precursor E3 lost 19 amino acid residues of its 35-amino acid leader sequence presumably due to a proteolytic cleavage. The recombinant mature E3 displayed comparable kinetic properties to those reported for highly purified mammalian E3s. The truncated precursor E3 showed about half of the mature E3 activity. The double-reciprocal plot for the mature E3 in the direction of NAD+ reduction showed parallel lines (ping-pong mechanism) while that for the truncated precursor E3 displayed intersecting lines (sequential mechanism). In the direction of NADH oxidation, the kinetic mechanisms of both E3s were apparently a ping-pong mechanism. These kinetic results showed that the partial 16-amino acid extension in the leader sequence changed the kinetic mechanism of human E3 so that it resembled that of glutathione reductase.     

Expression of human angiotensinogen cDNA in Escherichia coli

Human angiotensinogen cDNA clones were isolated from a human liver library. Nucleotide sequence analysis of these cDNA clones revealed that position 1075 in the messenger RNA, which is part of a PstI recognition sequence, is different from the published sequence (Kageyama, R., Ohkubo, H., and Nakanishi, S. (1984) Biochemistry 23, 3603-3609). This change results in an altered amino acid at this position in the corresponding protein sequence and suggests possible restriction fragment length polymorphism. The full length human angiotensinogen cDNA was constructed from partial cDNA clones and ligated into an isopropyl-1-thio-beta-D-galactopyranoside inducible bacterial expression vector pUC9 to develop expression plasmid pUCHAG27. This plasmid permitted the synthesis of human angiotensinogen in Escherichia coli. The recombinant bacteria overproduced a 53-kDa protein which was recognized by anti-human angiotensinogen antibodies. The synthesis of this protein was greatly increased upon induction with isopropyl-1-thio-beta-D-galactopyranoside. The chimeric protein, almost identical to human angiotensinogen, was partially purified by ammonium sulfate fractionation and gel filtration on Sephadex G-100. Human kidney renin was shown to enzymatically cleave this recombinant protein to produce des-(angiotensin I)-angiotensinogen and a small polypeptide. Thus, we provide evidence that recombinant human angiotensinogen synthesized through E. coli is biologically active and serves as a substrate for human renin.     

Signal sequence of preproglycinin affects production of the expressed protein in Escherichia coli

The effect of the signal peptide portion on the bacterial production of preproglycinin, a precursor of soybean storage protein, was examined. Nucleotide sequences corresponding to the signal peptide and the mature N-terminal region were deleted stepwise from the cDNA encoding the glycinin A1aB1b subunit precursor, and the deleted cDNAs were placed under the control of trc promoter in an expression vector pKK233-2. When the amounts of the protein products in Escherichia coli from each expression plasmid were determined, no accumulation of preproglycinin was observed from the plasmids with the full length or the five amino acids of the signal sequence. However, significant accumulation of the preproglycinin homologue proteins was noted from the plasmids retaining less than three amino acids of the signal sequence depending on the extent of deletion. N-terminal amino acid sequences of the products coincided with those predicted from the deleted cDNAs. The preproglycinin homologue proteins expressed from the mutant plasmids assembled into trimers of about 8S.     

人载脂蛋白AⅠ基因的克隆表达及功能

To identify, clone ,sequence and highly express the mature peptide gene of ApoA Ⅰ, total RNA was prepared from human fetal liver tissue. cDNA fragment encoding human ApoA Ⅰ was amplified by RT-PCR using specific primers, and then was inserted in pGEM-T vector. DNA sequencing indicates that the fragment is 729 base pairs in length and has 100% nucleotide homology with that of reported ApoA Ⅰ cDNA gene previously. The ApoA Ⅰ gene was cloned into pGEX 5X-1.The recombinant protein was expressed in E.coli DH5α, purified by glutathione-Sepharose 4B affinity chromatography and confirmed by SDS-PAGE. It was shown that the recombinant ApoA Ⅰ was expressed in E.coli, and the target protein amounted to 36% of total bacteria proteins. Cholesteryl ester transfer experiment showed that the recombinant ApoA Ⅰ was capable of promoting transfer of CE from HDL to LDL. Western blotting showed that the protein could react specifically with anti-ApoA Ⅰ antibodies.     

人β2-微球蛋白基因克隆及其在大肠杆菌中的高效表达

β2-微球蛋白(β2m)是主要组织相容性复合体(MHC)Ⅰ类分子的轻链部分,为制备MHCⅠ类分子四聚体的必要成分。根据已报道的序列设计特异引物,利用RT-PCR方法从人白细胞中克隆了β2m基因,并构建了成熟β2m的原核表达载体,在大肠杆菌中得到高效表达。表达的β2m大部分在包涵体中,经洗涤、变性和复性,并以强阴离子交换柱层析纯化,获得SDS-PAGE纯的人重组β2m,Western印迹法分析表明该蛋白具有与抗人天然β2m抗体反应的特性。此工作为制备MHCⅠ类分子四聚体奠定基础。     

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

Cloning, characterization, and expression of the beta subunit of pig heart succinyl-CoA synthetase.

The form of succinyl-CoA synthetase found in mammalian mitochondria is known to be an alpha beta dimer. Both GTP- and ATP-specific isozymes are present in various tissues. We have isolated essentially identical complementary DNA clones encoding the beta subunit of pig heart succinyl-CoA synthetase from both newborn and adult tissues. These cDNAs include a 1.4-kb sequence encoding the cytoplasmic precursor to the beta subunit comprised of 417 amino acid residues including a 22-residue mitochondrial targeting sequence. The cDNA encoding the 395-amino acid, 42,502-Da mature protein was confirmed to be the succinyl-CoA synthetase beta subunit by agreement with the N-terminal protein sequence and by high homology to prokaryotic forms of the beta subunit that were previously cloned (about 45% identical to beta from Escherichia coli). In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem. J. 250, 429-434), we found no tryptophan residue to be encoded in the sequence for the mature beta subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl-CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA clones encoding the mature pig heart beta subunit and its counterpart alpha subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl-CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.     

日本蟳高血糖激素基因的克隆与表达分析

通过RACE技术克隆获得日本蟳Charybdis japonica高血糖激素基因（CjCHH）全长cDNA序列;运用实时荧光定量PCR（qRT-PCR）方法分析该基因的组织差异性表达;利用原核表达技术获得CjCHH重组蛋白。序列分析表明：CjCHH cDNA全长1754 bp,包含111bp的5’末端非编码区（UTR）,423 bp的开放阅读框（ORF）,以及1236 bp的3’UTR,该基因可编码140个氨基酸。序列比对结果显示：CjCHH的成熟肽序列与其它甲壳动物CHH的一致性为41%~88%。系统进化树显示：CjCHH与其它梭子蟹科的CHH聚在一起,这与日本蟳所处的分类地位一致。组织差异性表达研究显示：CjCHH在检测的10个组织中均有表达,其中眼柄中表达量最高,肠和Y-器次之,其余组织表达量较低。成功构建了CjCHH重组表达质粒pET-CHH,并在大肠杆菌（Escherichia coli）中获得了高效表达,重组蛋白的相对分子量约11 kDa,与预测的相对分子质量大小相一致,表达水平在5 h的IPTG诱导过程中呈现上升趋势。     

Bacterial expression,purification, and characterization of rat kidney-type mitochondrial glutaminase

The human gene that encodes the kidney-type glutaminase (KGA) spans 84-kb, contains 19 exons, and encodes two alternatively spliced mRNAs. Various segments of the rat KGA cDNA were PCR amplified and cloned into a bacterial expression vector to determine whether the N- and C- terminal ends of the glutaminase protein were essential for activity. A recombinant glutaminase, lacking the coding sequence contained in exon 1, was found to be fully active. In contrast, proteins that lacked sequences from exons 1 and 2 and exons 1-3 were inactive. An additional construct that corresponded to the sequence encoded by exons 2-14 also retained full activity. Both of the fully active, truncated proteins were purified to apparent homogeneity using an incorporated N-terminal His(6)-tag and Ni(2+)-affinity chromatography. The K(M) values for glutamine of the native and recombinant forms of glutaminase were nearly identical. However, the two truncated forms of the glutaminase exhibit the characteristic phosphate activation profile only when dialyzed into a buffer lacking phosphate. Dialysis versus 10mM Tris-phosphate was sufficient to form an active tetramer. Thus, the deleted N-terminal sequence may contribute to the phosphate-dependent oligomerization and activation of the native glutaminase.     

In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin

Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% alpha-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 microg/ml. Refolded His-rMcnapin exhibited approximately 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.     

Presence in the stroma of chloroplasts of a large pool of a ribosomal protein not structurally related to any Escherichia coli ribosomal protein

Summary A search was made for the presence of a pool of free ribosomal proteins in the stroma of the spinach chloroplast. The results showed that a relatively large amount of one protein, CS-S5, is present in the stroma. Immunoprecipitation experiments showed that this protein is encoded by the nuclear genome. Clones were isolated from a cDNA library constructed in the expression vector lambda gtll, using specific antibodies raised against the CS-S5 protein. A full-length cDNA was sequenced which contains an open reading frame (ORF) for the precursor of the CS-S5 protein, as shown by immunoprecipitation. This precursor contains a putative transit peptide of 66 amino acids and the mature product has no significant homology with any of the Escherichia coli ribosomal proteins, in contrast to the other ribosomal protein gene products so far identified in spinach chloroplasts.     

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.     

Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40

We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote.