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November 23 2017

CandyTon

Part One: Will Phagotherapy Be a New Nemesis for Antibacterial Antibiotics?

Make the Immune Responses of Microorganism Engineered with CRISPR Attack Itself

Genetic modification of virus can lead to bacterial "suicide", which may be the next fight against antibiotic-resistant infections.

According to a report at the 2017 CRISPR Conference held in Montana, USA, several companies have used CRISPR gene editing system to engineer such viruses which were called phages, in order to kill specific types of bacteria. These companies will start clinical trials as early as next year.

Rodolphe Barrangou, chief scientific officer of North Carolina’s Locus Biosciences, said at the conference that preliminary tests showed that these phages rescued the mice that would otherwise had died of antibiotic-resistant infections.

Phages, isolated and purified from nature, have long been used to treat human infections, Eastern Europe in particular. These viruses infect only certain types of bacteria or bacterial strains, and therefore have less impact on the body's natural microbiome than antibiotics. It is generally accepted that they can be used on human beings, and it is totally safe.

However, phagotherapy has been developing slowly. Part of the reason is that these viruses exist naturally and cannot be patented. Bacteria can also rapidly evolve resistance to native phages, which means that researchers must continually isolate new phage that can fight against the same bacterial strain or bacterium. For regulators, it is harder to implement new treatments continuously.

Use CRISPR to Drive Bacterial Death

To avoid these problems, Locus and several other companies are working on phage that will allow the bacterial immune system CRISPR to target itself. Locus's phage targets antibiotic-resistant bacteria whose CRISPR system contains the DNA that can give instructions for a modified guide RNA, which locks on to parts of an antibiotic-resistant gene. Once a phage is infected with a bacterium, the guide RNA "grabs" its resistance gene. This triggers the Cas3 enzyme to destroy the gene sequence, which bacteria normally produce to kill the phage. Eventually, Cas3 destroys all the DNAs and kills the bacteria. "I now find it a bit ironical to use phages to kill bacteria like this," said Barrangou.

Eligo Bioscience, another Paris-based company, also uses a similar approach. It removes all the genetic instructions that allow phage replication and inserts DNA that codes for the guide RNA and the bacterial enzyme Cas9. Cas9 cuts bacterial DNA at the designated site, causing the bacteria to self-destruct. Xavier Duportet, Eligo's chief executive, said that the system's future goal is human intestinal pathogens, but he declined to say exactly what the pathogens are.

About Creative Biolabs

Creative Biolabs is a biotech company located in New York. Since its establishment, Creative Biolabs has been providing custom biotechnology and pharmaceutical services that cover the full scope of biotechnology needs of early drug discovery and development to customers all over the world, such as immunized antibody library phage, scfv phage display library, antibody engineering, etc.

May 29 2015

CandyTon
CandyTon

The Updated Technique in De Novo Antibody Sequencing

Antibody Sequencing has been a crucial technique in researching and developing various antibodies. There are many manufacturers who are dedicated to make this technique much better. 

To differ themselves from the crowd, they have develop something unique, Creative Biolabs, for instance. Their antibody sequencing method is quite unusual.

De Novo  Sequencing of the CDR3 region

While the CDR3 of the light chain is mostly encoded by the germline sequences, the CDR3 of the heavy chain is usually not available in databases. It is encoded by the so called D-segments but these are modified by nucleases and terminal transferases. Typically, only 1-4 AA of a D-segment remain in the matured antibody. The rest of the D-segment is “artificial” and has to be sequenced de novo.

Creative Biolabs generates many overlapping peptides during the fragmentation process, enabling us to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows them to read the CDR3 like a book. The technique is so powerful that they were able to sequence a 20 kDa protein, which had no homologue in the database.

Isobaric amino acids 

In contrast to other MS based methods, they can discriminate most isobaric amino acid combinations as examples listed.
• W can be distinguished from GE, AD and SV (by mass difference)
• R can be distinguished from GV (by mass difference) 
• Q can be distinguished from GA and K (by fragment spectra and mass difference) 
• N can be distinguished from GG (by derivatization and fragment spectra) 
• Leucine and isoleucine cannot be distinguished. However, most of these positions can be determined using the corresponding germline sequence (see figure 1). As antigen binding is mostly mediated by salt bridges and hydrogen bonds the impact of Leu/Ile is usually negligible. 

Sequencing of fluorochrome mABs, IgMs and other non-standard antibodies

Their method is usually not affected by small ligands (FITC, Biotin, Alexa) coupled to antibodies. Larger protein ligands make sequencing slightly more difficult. However, this can be compensated by using a higher protein concentration, because sequencing quality is dependent on sample amount.

 IgMs can usually be sequenced like normal IgGs. Slightly more sample may be required. As the constant region is modified by several glycans, which cannot be cleaved by PNGase F, they can only guarantee complete coverage for the variable part of the antibody. Their method works best with mouse and rat antibodies. However, ~50 antibodies from rabbit hamster and lama have been sequenced successfully. Besides, many older hybridoma produce 2 light chains (one is a nonsense light chain). These mixtures can usually be sequenced, even when the nonsense chain is in twofold molar excess.

Learn more about unique de novo antibody sequencing.

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