Keyhole limpet hemocyanin
From DrugPedia: A Wikipedia for Drug discovery
(New page: '''Keyhole limpet hemocyanin''' category: CarboDB) |
(→References) |
||
| (3 intermediate revisions not shown.) | |||
| Line 1: | Line 1: | ||
'''Keyhole limpet hemocyanin''' | '''Keyhole limpet hemocyanin''' | ||
| + | Keyhole limpet hemocyanin (KLH) is a large, multisubunit, oxygen-carrying, metalloprotein found in the hemolymph of the giant keyhole limpet, Megathura crenulata, that lives off the coast of California from Monterey Bay to Isla Asuncion off Baja California. | ||
| + | ==Protein properties== | ||
| + | Keyhole limpet hemocyanin is an extremely large, heterogeneous glycosylated protein consisting of subunits with a molecular weight of 350,000 and 390,000 in aggregates with molecular weights of 4,500,000-13,000,000. Each domain of a KLH subunit contains two copper atoms that together bind a single oxygen molecule (O2). When oxygen is bound to hemocyanin, the molecule takes on a distinctive transparent, opalescent blue color. The KLH protein is potently immunogenic yet safe in humans and is therefore highly prized as a vaccine carrier protein. The large and highly glycosylated KLH protein cannot be reproduced synthetically. It is available only as a purified biological product from the Keyhole Limpet Megathura crenulata. | ||
| + | [edit]Purification | ||
| + | KLH is purified from the hemolymph of Megathura crenulata by a series of steps that typically includes ammonium sulfate precipitation and dialysis, and may involve chromatographic purification to obtain the highest purity. KLH purification may also include endotoxin removal, but this step is often unnecessary because the endotoxin serves as an adjuvant when injected for antibody production. | ||
| + | If the protein becomes denatured or if the copper ions are lost in the purification process, the opalescent blue color disappears and the solution becomes a dull grayish color. Denaturation of KLH also results in a tendency of the protein to aggregate and precipitate from solution. | ||
| + | ==Use in biotechnology== | ||
| - | [[category: | + | Keyhole limpet hemocyanin (KLH) is used extensively as a carrier protein in the production of antibodies for research, biotechnology and therapeutic applications. Haptens are substances with a low molecular weight such as peptides, small proteins and drug molecules that are generally not immunogenic and require the aid of a carrier protein to stimulate a response from the immune system in the form of antibody production. KLH is the most widely employed carrier proteins for this purpose. KLH is an effective carrier protein for several reasons. Its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues for coupling haptens, allows a high hapten:carrier protein ratio increasing the likelihood of generating hapten-specific antibodies. In addition, because KLH is derived from the limpet, a gastropod, it is phylogenetically distant from mammalian proteins, thus reducing false positives in immunologically based research techniques in mammalian model organisms. |
| + | KLH may also be a challenging molecule to work with because of its propensity to aggregate and precipitate. Aggregates remain immunogenic, but limit the ability to conjugate haptens and are difficult to manipulate in the laboratory. A high quality KLH preparation with the clear opalescent blue color is the best indicator of KLH solubility. | ||
| + | |||
| + | |||
| + | ==Hapten coupling== | ||
| + | |||
| + | Haptens can be coupled to KLH using several methods. A simple one-step coupling can be performed using the crosslinker EDC to covalently attach carboxyls to primary amines. This method is the simplest to perform and the "random" orientation allows for antibody generation against all possible epitopes, but it generally results in some degree of polymerization, which decreases solubility making the conjugate more difficult to handle. | ||
| + | KLH can be activated with the crosslinker Sulfo-SMCC, which converts lysine residues to sulfhydryl-reactive maleimide groups. A sulfhydryl-containing hapten can then be reacted with the KLH to complete the immunogen without causing polymerization. The specificity of this reaction is ideal for situations where the cysteine is located away from the desired epitope (e.g. in peptides where a terminal cysteine can be added to either end of the peptide). Maleimide activated KLH, where the first part of this two step procedure has been completed, is commercially available. | ||
| + | |||
| + | |||
| + | ==Other carrier proteins== | ||
| + | |||
| + | Concholepas concholepas hemocyanin (marketed as Blue Carrier) | ||
| + | Bovine serum albumin (BSA) | ||
| + | Cationized BSA (cBSA) | ||
| + | Ovalbumin | ||
| + | |||
| + | |||
| + | ==Use in cancer therapy== | ||
| + | |||
| + | KLH is being tested as a therapeutic vaccine for a variety of cancers, including non-Hodgkins lymphoma, cutaneous melanoma, breast and bladder cancer. These vaccines use specific tumor-associated antigens (Haptens) conjugated to KLH to stimulate the body’s immune system to generate anti-tumor immune responses which can destroy tumor cells. The KLH carrier protein is responsible for conferring immunogenicity to the tumor antigens in these vaccines The rapidly growing interest in therapeutic vaccines (i.e. active immunotherapies) for cancer and the documented efficacy of KLH as a superior carrier protein for cancer vaccines are creating a significant biopharmaceutical market for KLH formulations. | ||
| + | |||
| + | |||
| + | ==Ecological considerations== | ||
| + | |||
| + | Because of the beneficial uses of KLH, the Giant Keyhole Limpet, from which KLH is derived, has been a subject of growing concern among fisheries biologists in the state of California due to the possibility of overharvest of the species for commercial purposes. Giant Keyhole Limpets are not an abundant species to begin with, and some divers have reported a decline in their populations. In a 2006 report from the Marine Science Institute, University of California, Santa Barbara, scientists state that they are "very concerned about the emerging fishery" for Giant Keyhole Limpets and suggest the potential exists to "decimate their populations". To help assure a secure long-term source of KLH for the many Federally sponsored biomedical research programs using KLH, Federal agencies including the National Institutes of Health[8] and the National Science Foundation have also sponsored research to establish sustainable mariculture production of the Giant Keyhole Limpet. In light of the sensitivity of this species, one should consider the source of KLH and obtain KLH only from sources that use Limpets grown in mariculture rather than harvested from the small and sensitive wild populations. | ||
| + | |||
| + | |||
| + | ==References== | ||
| + | |||
| + | [http://en.wikipedia.org/wiki/Keyhole_limpet_hemocyanin Wikipedia] | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | [[category:PolysacDB]] | ||
Current revision
Keyhole limpet hemocyanin
Keyhole limpet hemocyanin (KLH) is a large, multisubunit, oxygen-carrying, metalloprotein found in the hemolymph of the giant keyhole limpet, Megathura crenulata, that lives off the coast of California from Monterey Bay to Isla Asuncion off Baja California.
Contents |
[edit] Protein properties
Keyhole limpet hemocyanin is an extremely large, heterogeneous glycosylated protein consisting of subunits with a molecular weight of 350,000 and 390,000 in aggregates with molecular weights of 4,500,000-13,000,000. Each domain of a KLH subunit contains two copper atoms that together bind a single oxygen molecule (O2). When oxygen is bound to hemocyanin, the molecule takes on a distinctive transparent, opalescent blue color. The KLH protein is potently immunogenic yet safe in humans and is therefore highly prized as a vaccine carrier protein. The large and highly glycosylated KLH protein cannot be reproduced synthetically. It is available only as a purified biological product from the Keyhole Limpet Megathura crenulata. [edit]Purification
KLH is purified from the hemolymph of Megathura crenulata by a series of steps that typically includes ammonium sulfate precipitation and dialysis, and may involve chromatographic purification to obtain the highest purity. KLH purification may also include endotoxin removal, but this step is often unnecessary because the endotoxin serves as an adjuvant when injected for antibody production. If the protein becomes denatured or if the copper ions are lost in the purification process, the opalescent blue color disappears and the solution becomes a dull grayish color. Denaturation of KLH also results in a tendency of the protein to aggregate and precipitate from solution.
[edit] Use in biotechnology
Keyhole limpet hemocyanin (KLH) is used extensively as a carrier protein in the production of antibodies for research, biotechnology and therapeutic applications. Haptens are substances with a low molecular weight such as peptides, small proteins and drug molecules that are generally not immunogenic and require the aid of a carrier protein to stimulate a response from the immune system in the form of antibody production. KLH is the most widely employed carrier proteins for this purpose. KLH is an effective carrier protein for several reasons. Its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues for coupling haptens, allows a high hapten:carrier protein ratio increasing the likelihood of generating hapten-specific antibodies. In addition, because KLH is derived from the limpet, a gastropod, it is phylogenetically distant from mammalian proteins, thus reducing false positives in immunologically based research techniques in mammalian model organisms. KLH may also be a challenging molecule to work with because of its propensity to aggregate and precipitate. Aggregates remain immunogenic, but limit the ability to conjugate haptens and are difficult to manipulate in the laboratory. A high quality KLH preparation with the clear opalescent blue color is the best indicator of KLH solubility.
[edit] Hapten coupling
Haptens can be coupled to KLH using several methods. A simple one-step coupling can be performed using the crosslinker EDC to covalently attach carboxyls to primary amines. This method is the simplest to perform and the "random" orientation allows for antibody generation against all possible epitopes, but it generally results in some degree of polymerization, which decreases solubility making the conjugate more difficult to handle. KLH can be activated with the crosslinker Sulfo-SMCC, which converts lysine residues to sulfhydryl-reactive maleimide groups. A sulfhydryl-containing hapten can then be reacted with the KLH to complete the immunogen without causing polymerization. The specificity of this reaction is ideal for situations where the cysteine is located away from the desired epitope (e.g. in peptides where a terminal cysteine can be added to either end of the peptide). Maleimide activated KLH, where the first part of this two step procedure has been completed, is commercially available.
[edit] Other carrier proteins
Concholepas concholepas hemocyanin (marketed as Blue Carrier) Bovine serum albumin (BSA) Cationized BSA (cBSA) Ovalbumin
[edit] Use in cancer therapy
KLH is being tested as a therapeutic vaccine for a variety of cancers, including non-Hodgkins lymphoma, cutaneous melanoma, breast and bladder cancer. These vaccines use specific tumor-associated antigens (Haptens) conjugated to KLH to stimulate the body’s immune system to generate anti-tumor immune responses which can destroy tumor cells. The KLH carrier protein is responsible for conferring immunogenicity to the tumor antigens in these vaccines The rapidly growing interest in therapeutic vaccines (i.e. active immunotherapies) for cancer and the documented efficacy of KLH as a superior carrier protein for cancer vaccines are creating a significant biopharmaceutical market for KLH formulations.
[edit] Ecological considerations
Because of the beneficial uses of KLH, the Giant Keyhole Limpet, from which KLH is derived, has been a subject of growing concern among fisheries biologists in the state of California due to the possibility of overharvest of the species for commercial purposes. Giant Keyhole Limpets are not an abundant species to begin with, and some divers have reported a decline in their populations. In a 2006 report from the Marine Science Institute, University of California, Santa Barbara, scientists state that they are "very concerned about the emerging fishery" for Giant Keyhole Limpets and suggest the potential exists to "decimate their populations". To help assure a secure long-term source of KLH for the many Federally sponsored biomedical research programs using KLH, Federal agencies including the National Institutes of Health[8] and the National Science Foundation have also sponsored research to establish sustainable mariculture production of the Giant Keyhole Limpet. In light of the sensitivity of this species, one should consider the source of KLH and obtain KLH only from sources that use Limpets grown in mariculture rather than harvested from the small and sensitive wild populations.
