scholarly journals Production of Chymotrypsin-Resistant Bacillus thuringiensis Cry2Aa1 δ-Endotoxin by Protein Engineering

1999 ◽  
Vol 65 (10) ◽  
pp. 4601-4605 ◽  
Author(s):  
Mongkon Audtho ◽  
Algimantas P. Valaitis ◽  
Oscar Alzate ◽  
Donald H. Dean
Keyword(s):  
Bacillus Thuringiensis ◽  
Major Protein ◽  
Cleavage Sites ◽  
Mutant Proteins ◽  
Protein Fragments ◽  
Protease Cleavage ◽  
Protease Cleavage Sites ◽  
Domain I ◽  
Active Fragment

ABSTRACT Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) fromBacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when125I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.

In vivo selection of protease cleavage sites from retrovirus display libraries

1998 ◽  
Vol 16 (10) ◽  
pp. 951-954 ◽  
Author(s):  
Christian J. Buchholz ◽  
Kah-Whye Peng ◽  
Frances J. Morling ◽  
Jie Zhang ◽  
Francois-Loic Cosset ◽  
...  
Keyword(s):  
Cleavage Sites ◽  
In Vivo Selection ◽  
Protease Cleavage ◽  
Protease Cleavage Sites ◽  
Selection Of

scholarly journals Human Immunodeficiency Virus Type 1 Protease Cleavage Site Mutations Associated with Protease Inhibitor Cross-Resistance Selected by Indinavir, Ritonavir, and/or Saquinavir

2001 ◽  
Vol 75 (2) ◽  
pp. 589-594 ◽  
Author(s):  
Hélène C. F. Côté ◽  
Zabrina L. Brumme ◽  
P. Richard Harrigan
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitor ◽  
Cleavage Site ◽  
Cross Resistance ◽  
Cleavage Sites ◽  
Protease Cleavage ◽  
Immunodeficiency Virus ◽  
Before And After ◽  
Protease Cleavage Sites

ABSTRACT We examined the prevalence of cleavage site mutations, both within and outside the gag region, in 28 protease inhibitor (PI) cross-resistant patients treated with indinavir, ritonavir, and/or saquinavir compared to control patients treated with reverse transcriptase inhibitors. Three human immunodeficiency virus protease cleavage sites within gag (p2/NC, NC/p1, and NC/TFP) showed considerable in vivo evolution before and after therapy with indinavir, ritonavir, and/or saquinavir. Another gag cleavage site (p1/p6 gag ) showed a trend compared to matched controls. The other eight recognized cleavage sites showed relatively little difference between PI-resistant cases and controls. An A→V substitution at the P2 position of the NC/p1 and NC/TFP cleavage sites was the most common (29%) change selected by the PIs used in this study.

scholarly journals Conservation in vivo of Protease Cleavage Sites in Antigenic Sites of Poliovirus

1987 ◽  
Vol 68 (7) ◽  
pp. 1857-1865 ◽  
Author(s):  
P. D. Minor ◽  
M. Ferguson ◽  
A. Phillips ◽  
D. I. Magrath ◽  
A. Huovilainen ◽  
...  
Keyword(s):  
Cleavage Sites ◽  
Protease Cleavage ◽  
Antigenic Sites ◽  
Protease Cleavage Sites

Therapeutic Delivery of Stromal Cell-Derived Factor-1 for Injury Repair

Nano LIFE ◽  
2016 ◽  
Vol 06 (01) ◽  
pp. 1530001
Author(s):  
Agnes Yeboah ◽  
Martin L. Yarmush ◽  
Francois Berthiaume
Keyword(s):  
Wound Repair ◽  
Stromal Cell ◽  
Cellular Level ◽  
Cleavage Sites ◽  
Delivery Methods ◽  
Protease Cleavage ◽  
Cord Injury ◽  
Protease Cleavage Sites ◽  
Stromal Cell Derived Factor

Stromal cell-derived growth factor-1 (SDF1) is a chemokine that is over-expressed at sites of injury and is believed to play an important role in wound repair. At the cellular level, SDF1 regulates the mobilization and trafficking of endothelial progenitors that originate in the bone marrow and functionally contribute to neovascularization and angiogenesis in the wound. Consequently, SDF1 is a potentially interesting therapeutic with the potential to enhance these processes in acute and chronic injuries that otherwise tend to heal poorly, such as spinal cord injury, stroke, myocardial infarction, diabetic skin wounds and acute burns. However, the therapeutic usefulness of SDF1, as many other similar peptide-based growth factors and chemokines, is severely limited due to its short in vivo half-life, as it is rapidly degraded by proteases, which are typically very abundant at the wound site. Several studies have reported methodologies to increase SDF1 in vivo stability by mutating the protease cleavage sites of the molecule. Another approach has been to incorporate the chemokine into biomaterials that shield it from degradation. Yet another approach would be to develop a system that is inherently stable and could be combined with these aforementioned strategies. For example, self-assembled nanoparticles could shield SDF1 (or one of its forms engineered to be more resistant to proteolysis) from proteolysis and then be incorporated into suitable biomaterials. Nanotechnology-based delivery systems have however been used to a very limited extent for SDF1. This paper aims to provide a summary of the various stabilization and delivery methods available for SDF1, some of which have been already used, as well as others that have been used with other bioactive peptides, but would be potentially applicable to SDF1.

scholarly journals In Vivo Selection of Protease Cleavage Sites by Using Chimeric Sindbis Virus Libraries

2000 ◽  
Vol 74 (22) ◽  
pp. 10563-10570 ◽  
Author(s):  
Laura Pacini ◽  
Alessandra Vitelli ◽  
Gessica Filocamo ◽  
Linda Bartholomew ◽  
Mirko Brunetti ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Random Sequence ◽  
Biochemical Properties ◽  
Cleavage Sites ◽  
Viral Genomes ◽  
Protease Cleavage ◽  
Gene Coding ◽  
Protease Cleavage Sites

ABSTRACT Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

scholarly journals A novel HIV vaccine targeting the protease cleavage sites

2017 ◽  
Vol 14 (1) ◽  
Author(s):  
Hongzhao Li ◽  
Robert W. Omange ◽  
Francis A. Plummer ◽  
Ma Luo
Keyword(s):  
Hiv Vaccine ◽  
Cleavage Sites ◽  
Protease Cleavage ◽  
Protease Cleavage Sites

scholarly journals Proteolytic Cleavage of Bovine Adenovirus 3-Encoded pVIII

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Amit Gaba ◽  
Lisanework Ayalew ◽  
Niraj Makadiya ◽  
Suresh Tikoo
Keyword(s):  
Cleavage Site ◽  
Virus Assembly ◽  
Proteolytic Cleavage ◽  
Efficient Production ◽  
Cleavage Sites ◽  
Bovine Adenovirus ◽  
Protease Cleavage Site ◽  
Protease Cleavage ◽  
Protease Cleavage Sites ◽  
Single Protease

ABSTRACT Proteolytic maturation involving cleavage of one nonstructural and six structural precursor proteins including pVIII by adenovirus protease is an important aspect of the adenovirus life cycle. The pVIII encoded by bovine adenovirus 3 (BAdV-3) is a protein of 216 amino acids and contains two potential protease cleavage sites. Here, we report that BAdV-3 pVIII is cleaved by adenovirus protease at both potential consensus protease cleavage sites. Usage of at least one cleavage site appears essential for the production of progeny BAdV-3 virions as glycine-to-alanine mutation of both protease cleavage sites appears lethal for the production of progeny virions. However, mutation of a single protease cleavage site of BAdV-3 pVIII significantly affects the efficient production of infectious progeny virions. Further analysis revealed no significant defect in endosome escape, genome replication, capsid formation, and virus assembly. Interestingly, cleavage of pVIII at both potential cleavage sites appears essential for the production of stable BAdV-3 virions as BAdV-3 expressing pVIII containing a glycine-to-alanine mutation of either of the potential cleavage sites is thermolabile, and this mutation leads to the production of noninfectious virions. IMPORTANCE Here, we demonstrated that the BAdV-3 adenovirus protease cleaves BAdV-3 pVIII at both potential protease cleavage sites. Although cleavage of pVIII at one of the two adenoviral protease cleavage sites is required for the production of progeny virions, the mutation of a single cleavage site of pVIII affects the efficient production of infectious progeny virions. Further analysis indicated that the mutation of a single protease cleavage site (glycine to alanine) of pVIII produces thermolabile virions, which leads to the production of noninfectious virions with disrupted capsids. We thus provide evidence about the requirement of proteolytic cleavage of pVIII for production of infectious progeny virions. We feel that our study has significantly advanced the understanding of the requirement of adenovirus protease cleavage of pVIII.

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