Cusabio Vaccinia virus Recombinant

Shortly after the World Health Assembly resolution recommending the cessation of smallpox vaccination, proposals were made to use recombinant vaccinia viruses for immunization against other infectious agents. 161, 162 The idea was to stably insert one or more genes from other pathogens into the vaccinia virus genome while maintaining the latter’s infectivity. Furthermore, the high capacity of the vaccinia virus recombinant for foreign DNA raised the possibility of polyvalent vaccines against multiple diseases.

In principle, recombinant vaccinia viruses would have many of the properties of live attenuated virus vaccines and would naturally present antigens to stimulate humoral immunity to native protein conformation as well as cell-mediated immunity. Such vaccines could also retain the familiar advantages of the smallpox vaccine: heat stability, low cost, ease of administration, and a scar as visible proof of vaccination. Although recombinant vaccinia viruses are still being investigated for human and veterinary vaccination, their great value for vaccine research has been widely recognized.

Purity: greater than 85% as determined by SDS-PAGE.

Destination names: CTSK

Uniprot No.: P18378

Research Area: Others

Alternative names: (K2 protein)

Species: Vaccinia Virus (Western Reserve strain) (VACV) (Vaccinia virus (WR strain))

Source: Baculovirus

Expression Region: 1-88aa

Mole Weight: 11.6 kDa

Protein Length: Total length

Tag Information: N-terminal 6xHis-tagged

Form: Liquid or Lyophilized Powder

Note: We will preferably ship the format we have in stock, however, if you have any special requirements for the format, please remark your requirement when placing the order, we will prepare according to your demand.

Buffer

If the dosage form is liquid, the default storage buffer is Tris/PBS based buffer, 5%-50% glycerol. If the administration form is a lyophilized powder, the buffer before lyophilization is a Tris/PBS-based buffer, 6% trehalose, pH 8.0.

Reconstitution

We recommend that this vial be briefly centrifuged before opening to bring the contents to the bottom. Reconstitute protein in sterile deionized water at a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and an aliquot for long-term storage at -20°C/-80°C. Our final default glycerol concentration is 50%. Customers could use it for reference.

Storage Conditions

Store at -20°C/-80°C upon receipt, need to be aliquoted for multiple uses. Avoid repeated cycles of freezing and thawing.

Shelf life

Shelf life is related to many factors, storage condition, buffer ingredients, storage temperature and the stability of the protein itself. Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.

Delivery time

The delivery time may differ depending on the way or location of purchase, consult your local distributors for the specific delivery time.

Notes: Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.

Vaccinia virus as a tool for vaccine research

Recombinant vaccinia viruses provide a powerful means of dissecting the immune responses of humans and experimental animals to individual gene products of infectious agents. Only a few examples can be mentioned here. Therefore, recombinant vaccinia viruses were used to demonstrate that influenza virus HA and NP proteins induced subtype-specific and cross-reactive cytotoxic T-cell responses, respectively. Evidence for human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T cells in patients with acquired immunodeficiency syndrome (AIDS) was first obtained by using recombinant vaccinia viruses expressing envelope or internal proteins to prepare target cells.

Indeed, recombinant vaccinia viruses have become an important tool for cellular immunologists. Because proteins expressed in mammalian cells by recombinant vaccinia viruses are normally folded, processed, and transported, they can be used to induce or bind antibodies that recognize conformational epitopes. The wide host range of vaccinia viruses makes it possible to determine protective immune responses against infectious agents in a variety of experimental animals, from rodents to primates.

For example, the F glycoprotein is more important for inducing protection against respiratory syncytial virus, while the HN protein is better for parainfluenza virus type 3 and type 5. Protection elicited by the virus’s M2 protein syncytial is due to CD8+ T cells, while that induced by F and G proteins is due to antibodies. Similar results, with respect to HA and NP proteins, have been obtained in studies with the influenza virus. In some cases, vaccination has a priming effect followed by an anamnestic antibody response, as suggested for the protection of chimpanzees after inoculation with a recombinant vaccinia virus expressing hepatitis B surface antigen. A list of viruses for which protective immune responses have been elicited can be found in a review.

Cusabio Plasmodium falciparum Recombinant

Introduction:

The production of recombinant proteins is essential for the characterization and functional study of Plasmodium falciparum proteins. However, Plasmodium falciparum Recombinant proteins are among the most difficult to express, and when the expression is achieved, recombinant proteins generally misfold leading to inclusion body formation.

Objective:

Obtain and purify four recombinant proteins and use them as antigens to produce polyclonal antibodies. Production efficiency and solubility were evaluated as proteins were expressed in two Escherichia coli strains genetically modified to favour heterologous protein production (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).

Materials and methods:

The four recombinant proteins of P. falciparum correspond to the partial sequences of PfMyoA (Myosin A) and PfGAP50 (slip-associated protein 50) and the complete sequences of PfMTIP (a protein that interacts with the myosin tail). and PfGAP45 (slip-associated protein 45), were produced as glutathione S-transferase fusion proteins, purified and used to immunize mice.

  • Parasites

Parasite strains P. falciparum FCR3S1.2 and TM284S2 were cultured according to standard methods with 10% AB+ Rh+ serum added to the buffered medium (RPMI supplemented with Hepes, gentamicin and sodium bicarbonate). Genomic DNA from these parasites was purified using the EasyDNA purification kit (Invitrogen) according to the manufacturer’s protocol.

  • Recombinant plasmids

Plasmid constructs for expression of the recombinant proteins GST-DBL1α and GST-CIDR1α of FCR3S1.2var 1PfEMP1, GST-DBL1α and GST-DBL2β of TM284S2var 1PfEMP1 were generated as described previously [18, 19].

  • Codon optimization and gene resynthesis

The sequence of the DBL1α domain of FCR3S1.2var 1PfEMP1 was optimized for codon matching in E. coli. Genes were chemically resynthesized (GeneArt, Germany). Resynthesized DBL1α was amplified with oligonucleotide primers (BL-1 5′-ATG GCT ACT TCC GGA GGA, BL-1.1 5′-TTC GAT AAG CAG AAG AAG TAC) and cloned into pGEX4T-1 vector.

  • E. coli strain

The BL21-CodonPlus-RIL strain purchased from Stratagene (California, USA) was used for protein expression. This bacterial strain has been engineered to contain a high copy number of arginine U, leucine W, and isoleucine Y tRNA genes for optimal expression of heterologous proteins from organisms with A/T-rich genetic sequences.

  • Expression of PfEMP1 domains in BL21-CodonPlus-RIL bacteria

Competent BL21 cells were transformed with recombinant pGEX4T-1 plasmids containing FCR3S1.2 DBL1α, TM284S2 DBL1α or TM284S2 DBL2β as inserts. Transformed bacteria were selected on LB agar plates containing ampicillin (100 µg/ml). A single colony of transformed bacteria was inoculated into 30 ml of LB medium containing ampicillin (100 µg/ml) and chloramphenicol (50 µg/ml) for growth at 37°C overnight. Aliquots of the culture were inoculated into one litre of LB medium containing ampicillin (100 µg/ml).

The cultivation was carried out with a stirring speed of 225 rpm. The pH value and the optical density at A600 of the cultures were systematically controlled. Aliquots (50 ml) of each culture were taken sequentially after the OD A600 reached 0.5 and IPTG (isopropyl-b-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM to induce expression. Expression was carried out for three hours at 37°C and the bacteria were subsequently harvested by centrifugation at 4000 rpm for 15 minutes. Recombinant proteins were purified on glutathione-sepharose (Amersham-Pharmacia, Sweden).

  • SDS-PAGE analysis of recombinant proteins

To analyze the recombinant proteins, aliquots of the soluble and insoluble fractions of the expressed proteins from each purification were mixed with an equal volume of SDS-PAGE loading buffer containing β-mercaptoethanol and boiled at 100 °C for 5 min. Denatured proteins were resolved on 10% acrylamide gels containing 1% SDS and visualized by Coomassie brilliant blue solution staining.

  • Binding to heparin and blood group A antigen

Recombinant DBL1α purified from FCR3S1.2 expressed with pGEX plasmids containing the wild-type DBL1 sequence or the codon-optimized sequence was further passed through a HiTrap-heparin column (Amersham-Pharmacia Biotech, Sweden). After washing with PBS tween-20 buffer, bound protein was released from the column with 2M NaCl and immediately dialyzed against cold PBS. Aliquots of the eluted proteins were subjected to SDS-PAGE. The binding of recombinant FCR3S1.2 DBL1α to blood group A antigen was studied using a solid phase assay system.

Results:

Protein expression was much more efficient in BL21-CodonPlus, the strain containing tRNAs that are rare in wild-type E. coli, compared to expression in BL21-pG-KJE8. Although BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize inclusion body formation.

Conclusion:

The use of genetically modified E. coli strains was essential to achieve high levels of expression of the four P. falciparum proteins evaluated and improve the solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in sufficient quantity to produce polyclonal antibodies in mice and a good quantity of two pure and soluble recombinant proteins for future tests.

Keywords: Escherichia coli.; Plasmodium falciparum; recombinant proteins.

Cusabio Pongo abelii Recombinant

They are extremely intelligent and have shown evidence of tool use and culture, traits once believed to be uniquely human. Despite being one of our closest relatives, human activities are having a devastating impact on the Sumatran orangutan and its habitat. They are the slowest reproducing of all mammals, with mothers caring for their young for up to 7 years. With such a low reproductive rate, even a small decrease in numbers can lead to extinction.

There are only two species in its genus Pongo abelii Recombinant, the other being the Bornean orangutan. They show more tool use than their sister species from Borneo; compiling a ‘toolbox’ about their lives. Its main threats include the destruction of its lowland rainforest habitat for palm oil plantations and agriculture, logging, the creation of new roads, as well as the death of some by humans or the illegal pet trade. Concerted conservation efforts are needed to prevent this peaceful primate from becoming the first great ape to become extinct in the wild.

Purity: >85% (SDS-PAGE)

Destination Names: PIC

Uniprot No.: Q5RBP8

Alternative Names: PFCs; appropriatedine; P-factor complement

Species: Pongo abelii (Sumatran Orangutan) (Pongo pygmaeus abelii)

Expression Region: 28-469

Protein length: Total length of the mature protein

Label information

The following labels are available.

  • N-terminus His-tagged
  • Without tags
  • The type of label will be determined during the production process. If you have specified a tag type, let us know and we will develop the specified tag preferentially.

Form: Lyophilized powder

Buffer before lyophilization: Tris/PBS based buffer, 6% trehalose, pH 8.0

Reconstitution

We recommend that this vial be briefly centrifuged before opening to bring the contents to the bottom. Reconstitute protein in sterile deionized water at a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and an aliquot for long-term storage at -20℃/-80℃. Our final default glycerol concentration is 50%. Customers could use it for reference.

Storage Conditions

Store at -20°C/-80°C upon receipt, need to be aliquoted for multiple uses. Avoid repeated cycles of freezing and thawing.

Shelf life

Shelf life is related to many factors, storage condition, buffer ingredients, storage temperature and the stability of the protein itself. Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.

Delivery time

The delivery time may differ depending on the way or location of purchase, consult your local distributors for the specific delivery time.

Note: All of our proteins are shipped with regular blue ice packs by default. If you request shipping with dry ice, please contact us in advance and additional fees will be charged.

Notes:

Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.

How are they recognized?

Orangutans are commonly called “red apes” as they have long, soft orange hair. His skin is dark grey. There is a difference in height, which varies from 1.30 m to 1.80 m, but the big difference is in weight, which ranges from 30 to 90 kg. An adult male can be three times as heavy as an adult female.

Orangutans are the largest arboreal animals in the world. As they mainly live high up in the trees, they have developed long arms (up to 2.25m) to help them swing through the forest. Both their feet and hands are incredibly dexterous and they have opposable thumbs like we do, making it easy to pick and peel fruit.