Keyhole limpet hemocyanin: Difference between revisions
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'''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]] |
'''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]][http://www.enature.com/fieldguides/detail.asp?recnum=SS0048][http://www.starthrower.org/products/DDDB/DDDB_050-099/DDDB_084%20giant%20keyhole%20limpet.htm]. |
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===Protein properties=== |
===Protein properties=== |
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===Use in biotechnology=== |
===Use in biotechnology=== |
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Keyhole limpet hemocyanin 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 [[peptide]]s, 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. |
Keyhole limpet hemocyanin 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 [[peptide]]s, 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[http://jbt.abrf.org/cgi/content/full/18/3/173]. KLH is the most widely employed carrier proteins for this purpose. |
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KLH is an effective carrier protein for several reasons. It's large size and distinct/foreign [[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. |
KLH is an effective carrier protein for several reasons. It's large size and distinct/foreign [[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. |
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===Use in cancer therapy=== |
===Use in cancer therapy=== |
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KLH is being tested as a [[therapeutic]] [[vaccine]] for a variety of cancers, including non-Hodgkins [[lymphoma]], cutaneous [[melanoma]], breast and bladder |
KLH is being tested as a [[therapeutic]] [[vaccine]] for a variety of cancers, including non-Hodgkins [[lymphoma]], cutaneous [[melanoma]], breast and bladder cancer[http://www.cancer.gov/cancertopics/factsheet/cancervaccine]. |
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===Ecological considerations=== |
===Ecological considerations=== |
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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 [http://repositories.cdlib.org/cgi/viewcontent.cgi?article=1013&context=csgc], and some divers have reported a decline in their populations [http://www.starthrower.org/products/DDDB/DDDB_050-099/DDDB_084%20giant%20keyhole%20limpet.htm]. 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" |
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 [http://repositories.cdlib.org/cgi/viewcontent.cgi?article=1013&context=csgc], and some divers have reported a decline in their populations [http://www.starthrower.org/products/DDDB/DDDB_050-099/DDDB_084%20giant%20keyhole%20limpet.htm]. 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"[http://channelislands.noaa.gov/sac/pdf/rap_1-06.pdf]. 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. |
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[[Category:Proteins]] |
[[Category:Proteins]] |
Revision as of 18:27, 20 May 2008
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[1][2].
Protein properties
Keyhole limpet hemocyanin is an extremely large, heterogeneous 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.
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
Keyhole limpet hemocyanin 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[3]. KLH is the most widely employed carrier proteins for this purpose. KLH is an effective carrier protein for several reasons. It's large size and distinct/foreign 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.
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[4].
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 [5], and some divers have reported a decline in their populations [6]. 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"[7]. 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.