Monitoring global gene expression of proteases and improvement of human lysozyme production in the nptB gene disruptant of Aspergillus oryzae

Aspergillus oryzae has numerous protease genes that might cause proteolytic degradation of heterologously-produced proteins. The productivity of the heterologous protein can be improved by protease gene disruption, but it is difficult to select disruption targets efficiently. In this study, we monitored the expression of 132 protease genes by DNA microarray. A group of protease genes up-regulated during cultivation was identified by clustering analysis. In this protease group, the nptB gene encoding neutral protease II was included as well as the alpA, tppA, and pepA genes, disruption of which has improved human lysozyme (HLY) production. The nptB gene was disrupted to investigate its involvement in HLY production, and nptB disruptants showed an improvement in the production. These observations suggest that monitoring the expression of protease genes is an efficient strategy in screening potential disruption targets for heterologous protein production in A. oryzae.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli

For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.     

Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe

The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83–99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.     

Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism.

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.     

Regulation of expression of genes encoding digestive proteases in the gut of a polyphagous lepidopteran larva in response to dietary protease inhibitors

Abstract. Larvae of Helicoverpa armigera (Hübner), a polyphagous lepidopteran crop pest, adapt to the presence of protease inhibitors in their diet by differential regulation of multiple genes encoding digestive proteases. The time‐course of their response to dietary soybean Kunitz trypsin inhibitor (SKTI) involves several stages; an initial up‐regulation of all protease genes assayed (up to 12 h after exposure to inhibitor) is succeeded by a longer‐term down‐regulation of expression of specific genes that encode proteases most sensitive to the inhibitor, whereas genes encoding putative inhibitor‐insensitive proteases continue to be up‐regulated (after 24 h of exposure). Consequently, insect protease activity changes from being sensitive to the inhibitor, to being largely insensitive. The insect response is comparable in its timescale with that of the synthesis of protease inhibitors in the plant wounding response. SKTl causes similar effects on protease gene expression and gut protease activity when fed in diets containing casein or hydrolysed casein as sources of amino acid, suggesting that the insect response is not mediated through inhibition of digestive proteolysis. Soybean Bowman–Birk inhibitor, which has a broader range of inhibitory activity against gut proteases in H. armigera, but is a less effective inhibitor on an I₅₀ basis, proves to be a more effective antimetabolite than SKTI, but does not induce inhibitor‐insensitive protease activity because it causes a general up‐regulation of protease‐encoding genes. A possible mechanism to account for these different responses is discussed.     

Evolution of soldier-specific venomous protease in social aphids

In social aphids of the genus Tuberaphis a cysteine protease gene of the family cathepsin B exhibits soldier-specific expression and intestinal protease production. The product is orally excreted and injected by soldier nymphs into natural enemies, thereby exerting an insecticidal activity. In an attempt to gain insights into when and how the novel venomous protease for the altruistic caste has evolved, we investigated the soldier-specific type (S-type) and nonspecific type (N-type) cathepsin B genes from social and nonsocial aphids. All the social aphids examined, representing the genera Tuberaphis, Astegopteryx, and Cerataphis, possessed both the S-type and N-type genes. Phylogenetically distant nonsocial aphids also possessed cathepsin B genes allied to the S-type and the N-type, indicating the evolutionary origin of these genes in the common ancestor of extant aphids. In Tuberaphis species the S-type genes exhibited significant soldier-specific expression and accelerated molecular evolution whereas the N-type genes did not. In Astegopteryx and Cerataphis species, meanwhile, both the S-type and N-type genes exhibited neither remarkable soldier-specific expression nor accelerated molecular evolution. These results suggest that the S-type gene acquired the soldier-specific expression and the venom function after divergence of the genus Tuberaphis. On the structural model of the S-type protease of Tuberaphis styraci the accelerated molecular evolution was associated with the molecular surface rather than the catalytic cleft, suggesting that the venom activity was probably acquired by relatively minor modifications on the molecular surface rather than by generation of a novel active site. In Cerataphis jamuritsu the S-type gene was, although containing a stop codon, structurally almost intact and still transcribed, suggesting recent pseudogenization of the gene copy and possible relevance to relaxed functional constraint in the highly multiplied protease gene family. On the basis of these results we suggest that the massive amplification in aphid cathepsin B genes might have predisposed the evolution of venomous protease in the social aphid lineage and argue that gene duplication, accelerated molecular evolution, and acquisition of novel gene function must have played considerable roles in the evolution of complex biological systems including insect sociality.     

枯草芽孢杆菌中一种新型双向启动子的功能鉴定

本研究旨在通过转录组分析预测的方法,由地衣芽孢杆菌中筛选获得一种新型双向启动子,鉴定其启动强度。以已知强组成型启动子pShuttle-09为对照,检测其对克劳氏芽孢杆菌碱性蛋白酶基因的表达活性。成功构建了3种重组碱性蛋白酶表达载体及对应的工程菌株。在新型启动子pLA和其反向启动子pLB调控转录下,克劳氏芽孢杆菌碱性蛋白酶表达活性达到164 U/mL和111 U/mL。结果表明,pLA的启动强度明显高于pShuttle-09和pLB,pLA启动子与pLB启动子均可表达碱性蛋白酶。从而为枯草芽孢杆菌表达系统中异源基因的表达提供一个新的方向,也为原核生物中共同表达两种基因提供了新的思路。     

Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region

The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.     

TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector

Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells. However, conventional approaches are often inefficient, nonstoichiometric and may limit the maximum number of genes that can be simultaneously expressed. We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses. This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self-cleaving the fusion protein into functional components. To demonstrate this method in E. coli cells, we analyzed the translation products using SDS-PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex. To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products. We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector. In summary, the efficient expression and separation of up to three genes was achieved in both E. coli and mammalian cells using a single TEV protease self-processing vector.