scholarly journals Expression of the Neutral Protease Gene from a Thermophilic Bacillus sp. BT1 Strain in Bacillus subtilis and Its Natural Host: Identification of a Functional Promoter

2000 ◽  
Vol 182 (14) ◽  
pp. 4104-4107 ◽  
Author(s):  
Branislav Večerek ◽  
Gerard Venema
Keyword(s):  
Bacillus Subtilis ◽  
Natural Host ◽  
Neutral Protease ◽  
Protease Gene ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Gene Open Reading Frame ◽  
Efficient Expression ◽  
Rapid Amplification ◽  
Host Identification

ABSTRACT The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5′ to the gene. In contrast, in heterologous B. subtilis this thermophilic nprpromoter does not function, and expression of the npr gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5′ to ORF X that is required for efficient expression of thenpr gene in Bacillus subtilis as verified by primer extension, reverse transcription-PCR, and 5′ rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic Bacillus sp. BT1 strain are different from those used in B. subtilis.

Anti-Tumor Effects of a Newly Cloned Serine Protease from the Marine Annelid, Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 183-186
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acids ◽  
Serine Protease ◽  
Protease Gene ◽  
Mature Peptide ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Arenicola Cristata ◽  
Serine Protease Family ◽  
Rapid Amplification ◽  
Serine Protease Gene

We cloned a new serine protease gene from the marine annelid,Arenicola cristataby rapid amplification of cDNA ends. The full-length cDNA of 901bp contained an open reading frame of 774bp encoding 258 amino acids. Sequence analysis of the deduced amino acids indicated that this protease belonged to serine protease family and contained highly conserved sequence GDSGGP. An expression vector, harboring the mature peptide ofArenicola cristataprotease, was constructed and transformed intoE.coli. The purified recombinant protein could inhibit proliferation of cancer cells in a dose-dependant way and induce apoptosis. These results indicated that the recombinant protease ofArenicola cristata, as a new member of serine protease family, might be valuable in developing anti-tumor agents.

scholarly journals Subcloning and Expression of a Protease Gene from Bacillus halodurans CM1 in Bacillus subtilis DB104

2019 ◽  
Vol 16 (04) ◽  
pp. 817-826
Author(s):  
Endang Rahmawati ◽  
Abinawanto Abinawanto ◽  
Is Helianti
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Open Reading Frame ◽  
Bacillus Halodurans ◽  
Protease Gene ◽  
Erythromycin Resistance ◽  
Reading Frame ◽  
E Coli ◽  
Protease Enzyme ◽  
Ph Range

ABSTRACT: Proteases are potential enzymes that utilized in various industrial fields, and the demand of these enzymes is increasing. Bacillus halodurans CM1 is Indonesia indigenous bacterium which is detected to be able to produce alkalotermophilic protease enzyme. In this study, we subcloned the protease gene consist of Open Reading Frame of protease gene and its promoter from Bacillus halodurans CM1 in Bacillus subtilis DB104 via conjugation, and analyzed the expression of the recombinant protease. The protease gene is 1 417 bp length including the open reading frame and the promoter, and obtained by PCR and cloned into pGEM T easy. After confirmed by sequencing, the gene was subcloned into vector pBBRE194, then the recombinant plasmid was transformed into E. coli S17-1. This E.coli was then conjugated to Bacillus subtilis DB104. The target recombinant B. subtilis DB104 has been obtained confirmed by plasmid verification and erythromycin resistance. The recombinant protease produced showed the highest enzyme activity at 50oC and pH 9 (with pH range 5-9) which with protease activity 13.66 U/mL.

scholarly journals The SsrA-SmpB Ribosome Rescue System Is Important for Growth of Bacillus subtilis at Low and High Temperatures

2007 ◽  
Vol 189 (10) ◽  
pp. 3729-3737 ◽  
Author(s):  
Ji-Hyun Shin ◽  
Chester W. Price
Keyword(s):  
Bacillus Subtilis ◽  
High Temperature ◽  
Rnase R ◽  
Growth And Survival ◽  
Reverse Transcription Pcr ◽  
Growth Loss ◽  
Rescue System ◽  
Response Systems ◽  
Rapid Amplification

ABSTRACT Bacillus subtilis has multiple stress response systems whose integrated action promotes growth and survival under unfavorable conditions. Here we address the function and transcriptional organization of a five-gene cluster containing ssrA, previously known to be important for growth at high temperature because of the role of its tmRNA product in rescuing stalled ribosomes. Reverse transcription-PCR experiments detected a single message for the secG-yvaK-rnr-smpB-ssrA cluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR and lacZ fusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One σA-like promoter preceded secG (P1), and internal σA-like promoters were found in both the rnr-smpB (P2) and smpB-ssrA intervals (P3 and PHS). Another internal promoter lay in the secG-yvaK intercistronic region, and this activity (PB) was dependent on the general stress factor σB. Null mutations in the four genes downstream from PB were tested for their effects on growth. Loss of yvaK (carboxylesterase E) or rnr (RNase R) caused no obvious phenotype. By contrast, smpB was required for growth at high temperature (52°C), as anticipated if its product (a small ribosomal binding protein) is essential for tmRNA (ssrA) function. Notably, smpB and ssrA were also required for growth at low temperature (16°C), a phenotype not previously associated with tmRNA activity. These results extend the known high-temperature role of ssrA and indicate that the ribosome rescue system is important at both extremes of the B. subtilis temperature range.

scholarly journals Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

2014 ◽  
Vol 2014 ◽  
pp. 1-20 ◽  
Author(s):  
Norsyuhada Alias ◽  
Mu’adz Ahmad Mazian ◽  
Abu Bakar Salleh ◽  
Mahiran Basri ◽  
Raja Noor Zaliha Raja Abd. Rahman
Keyword(s):  
Signal Sequence ◽  
Alcohol Oxidase ◽  
Protease Production ◽  
Protease Gene ◽  
Gene Encoding ◽  
Reading Frame ◽  
Antarctic Yeast ◽  
Glaciozyma Antarctica Pi12 ◽  
Rapid Amplification ◽  
High Level

Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa.

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