EP1824974A2 - Procede pour recuperer des proteines a partir de larves d'insectes - Google Patents

Procede pour recuperer des proteines a partir de larves d'insectes

Info

Publication number
EP1824974A2
EP1824974A2 EP05821398A EP05821398A EP1824974A2 EP 1824974 A2 EP1824974 A2 EP 1824974A2 EP 05821398 A EP05821398 A EP 05821398A EP 05821398 A EP05821398 A EP 05821398A EP 1824974 A2 EP1824974 A2 EP 1824974A2
Authority
EP
European Patent Office
Prior art keywords
larvae
proteins
insect larvae
insect
extraction buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05821398A
Other languages
German (de)
English (en)
Inventor
Richard W Welch
Susan G Brown
James H Campbell
Stuart E Builder
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chesapeake Perl Inc
Original Assignee
Chesapeake Perl Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chesapeake Perl Inc filed Critical Chesapeake Perl Inc
Publication of EP1824974A2 publication Critical patent/EP1824974A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects

Definitions

  • the invention relates to a method and system for recovery of proteins from insect larvae. More particularly, the invention relates to recovery of proteins from mass reared insect larvae.
  • Insects have been used to mass produce both native and recombinant proteins.
  • One drawback to producing proteins utilizing insects relates to difficulties encountered in isolating proteins from the insect bodies, particularly the initial extraction of the proteins.
  • methods for recovery of proteins from insect larvae include manual extraction of the proteins.
  • Manual extraction typically involves the use of syringes to remove the hemolymph or removal of a portion of the insect body, such as severing abdominal legs, to then "bleed" the insect as the hemolymph drains out of the insect.
  • Manual extraction is very labor intensive and time consuming.
  • the process is not scalable and therefore suitable only for small numbers of insects. As a result, manual extraction is not useful for large scale production of significant quantities of protein.
  • the insect larvae are completely homogenized.
  • complete homogenization results in the generation of an extremely complex mix of intracellular and extracellular proteins, gut proteases, intracellular proteases, integumin, chitin, organ, and cellular debris, as well as the formation of substantial amounts of lipid micelles and multi- lamellar vesicles.
  • homogenization decreases the size of the overall volumetric solids without decreasing the overall percentage of volumetric solids, making recovery more difficult and costly. The desired proteins must then be isolated from this complex mixture.
  • fusion proteins combining the protein of interest with protein components of the silkworm cocoon are utilized for recovery of the protein of interest from the silkworm cocoon. Problems associated with this process include incorrect folding of the target proteins due to the additional amino acid sequence(s) from the cocoon protein(s) required to create the fusion protein, as well as problems associated with the removal of those same extra amino acid sequences comprising the components of the silkworm cocoon proteins from the protein of interest.
  • the present invention provides a method for recovery of proteins from insect larvae.
  • the method includes mixing in an extraction buffer at least one of whole insect larvae and non-homogenized parts of insect larvae.
  • the buffer is separated from the at least one of whole insect larvae and non-homogenized parts of insect larvae.
  • the proteins are isolated from the separated buffer.
  • the present invention also provides a system for recovery of proteins from insect larvae.
  • the system includes a mill to cut up the larvae into pieces, a mixing vessel operative to receive the larvae cut into pieces and an extraction buffer, a container operative to receive extraction buffer after mixing with the larvae cut into pieces, and a separator operative to separate the proteins from larval debris.
  • Fig. 1 is a flowchart showing elements of an embodiment of a process according to the present invention
  • Fig. 2 is a diagram of an embodiment of a system according to the present invention, illustrating process flow
  • Fig. 3 is a photograph that shows results of Example 1;
  • Fig. 4 is a photograph that shows results of Example 2.
  • Figs. 5 and 6 are graphs that illustrate results of Example 3.
  • Fig. 7 is a graph that shows results of Example 4.
  • Embodiments of the present invention provide a method and a system for extracting hemolymph from insects.
  • the present invention provides a method that may be applied to process large quantities of insects on an industrial scale. Once the hemo lymph is extracted, proteins and/or other desired components present in it may be isolated. By providing a method for large scale recovery of hemo lymph, the present invention helps to make practical protein production in insects.
  • the present invention is useful in a number of purposes for mass rearing of insects.
  • the present invention is useful in the mass rearing of insects to manufacture recombinant or wild-type baculo viruses.
  • Such baculoviruses may be utilized as bioinsecticides, such as baculoviruses, entomopathogenic fungi, or nematodes, for example.
  • insects may be mass reared to manufacture recombinant proteins using baculovirus-mediated expression.
  • the method of the present invention includes mixing whole insects, whole insect larvae, cut up whole insects and/or cut up insect larvae with an extraction buffer. If the insect and/or larvae are utilized, they are not homogenized or are minimally homogenized, meaning that the insect and/or larvae are not chopped so thoroughly that the resulting pieces are indistinguishable. When insects or larvae are chopped to complete homogenization, proteins that may be the target of the process can become mixed with other undesired components in the insects, such as non-secreted proteins, various proteases, chitin, integumin, whole cells, lipid bodies and other undesired insect products.
  • insects examples include, but are not limited to, Trichoplusia ni (cabbage looper), Spodoptera exigua (beet armyworm), Spodoptera frugiperda (fall armyworm), Heliothis virescens (tobacco budworm), Helicoverpa zea (bollworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Anagrapha falcifera (celery looper), Cydia pomonella (codling moth), Cryptophlebia leucotreta (false codling moth), larval stages of other moth species, butterfly species, predatory insects, parasitic insects, grasshoppers, crickets, katydids (Orthoptera), cockroaches (Blattodea), mantids, walking sticks, earwigs (Mantodea, Phasmatodea, Dermaptera),
  • Any protein produced utilizing any insect may be recovered.
  • proteins and protein classes that may be recovered include monoclonal antibodies, cell surface receptors, membrane transport proteins, cyclins, cytokines, Fab fragments, viral antigens, fluorescent proteins, fusion proteins, growth factors, cholinesterases, peptidases, alpha-interferon, murine IgG, porcine interleukm-18, human adenosine deaminase, human Group II Phospholipase A2, interleukin-2, viral receptors, hormonal receptors, invertebrate immune proteins, kinases, phosphatases, RAS effectors, viral antigens, and antimicrobial peptides.
  • Fig. 1 illustrates general steps involved in an embodiment of a method according to the invention for recovering protein(s) from insect larvae. Initially, the larvae may be commuted or cracked (step 1). This is optional step can depend upon the protein involved.
  • the larvae Prior to being cut, the larvae may be frozen. Freezing can help to minimize homogenization of the larvae.
  • the larvae may be chopped, fractured by impacting, such as with a hammer or mallet, milled or otherwise broken into pieces or comminuted. According to other embodiments, the insects are freeze dried.
  • Comminution of the larvae is performed to increase the flowability of the larvae by forming them into a granular mixture and to increase the surface area of the larvae, decrease the distance required for diffusion of the protein of interest, increase yield of proteins recovered from the larvae and/or provide other benefits.
  • the larvae are broken into pieces having dimensions of about 1 mm to about 1 cm.
  • the process may utilize pieces anywhere from whole larvae down to pieces about 50 microns.
  • Favorable results may be obtained with a granular having a rough diameter of about 0.5 mm to about 2.5 mm.
  • An alternative embodiment includes pressing the larvae into flakes with dimensions typically about 0.1 mm to about 5 mm and more typically about 0.5 mm to about 2 mm.
  • the larvae are chopped into granules on the order of about 2 mm.
  • the insects/larvae may be mass reared in a web that includes a plurality of wells formed therein and covered by a cover. Each well is filled with formulated insect diet and at least one insect egg. The insects are raised to a larval or beyond stage or beyond and may or may not be infected with a virus. If the insects are mass reared in such a manner, the web, the cover and the contents of the wells, including the insect diet and the insects may be chopped up and mixed with the extraction buffer. In such a case, the entire chopped contents may be mixed with extraction buffer.
  • the insect larvae are comminuted with a milling machine.
  • a particular milling machine that may be used is a Fitzmill milling machine.
  • the machine may be operated such that the blades face in the forward direction resulting in a granular form of the frozen larvae.
  • the mill may be operated at various speeds to result in cutting of the larvae to various degrees.
  • the mill could be operated at about 3000 to about 9000 rpm.
  • Specific embodiments have utilized about 3000, about 6000 or about 9000 rpm.
  • Comminutation may be carried out at reduced temperature.
  • the comminutation may be carried out at a temperature range of
  • the insect larvae are comminuted with a conical mill.
  • a particular milling machine that may be used is a Quadro Comil.
  • the mill may be operated at various speeds and with various configurations to result in cutting of the larvae to various degrees.
  • the mill could be operated at about 3000 to about 5000 rpm.
  • Specific embodiments have been utilized at about 3500 rpm.
  • Varying configurations for the screen and impeller include square and round, cutting and non-cutting and pore or opening sizes ranging from about 1 to about 15 mm.
  • Specific embodiments have utilized round and square impellers, cutting and non-cutting screens and pore sizes ranging from about 2 to about 9 mm.
  • the comminutation may be carried out at a temperature range of
  • RPM for communitation may depend on the blades used in the comminutation machinery and the orientation of the blades.
  • comminutation by impactation may be performed by reversal of the blades, which results in striking the larvae with the flat side of the blades, under extreme cold conditions.
  • the cold conditions are created by injecting the striking region of the equipment with vapor phase liquid nitrogen or carbon dioxide.
  • Comminutation by impacting may generally be performed by increasing the striking force, either by lengthening the striking arm or by increasing the rotation speed of the flat blade.
  • a third technique includes scaling the material to be sized through a "sieving" device or screen whole impacting. Either the blade or the flat of the striker may be oriented towards the larvae. This technique may be carried out at low temperatures and slower speeds. The size reduction may be controlled by scraping or dragging the larvae over the screen or sieve.
  • a fourth technique includes chopping the larvae by passage of the larvae through a series of rotating blades that cut the larvae on a cutting surface.
  • the pieces of larvae, whole larvae, whole insects and/or pieces of insects may then be mixed with an extraction buffer.
  • the extraction buffer may include any components in which the target protein(s) are soluble.
  • the buffer includes 50 mM Tris having a pH of 7.8, 150 niM NaCl, and 5 mM beta-mercaptoethanol.
  • the pH of the buffer could be about 5.0 to about 8.5.
  • buffer ingredients used in the extraction of target proteins or materials of interest include: citrate (1OmM to 1 M); Tris (10 mM to 1 M); phosphate (10 mM to 1 M); buffers from the Goode buffer series (10 mM to 1 M); acetate (10 mM to 1 M); glycerol (5% to 50%); PEG (0.1% to 10 %); ammonium sulfate ( 100 mM to 1.5 M); detergents, both ionic and non-ionic, such as Triton X-IOO, Tween 20 or Tween 80, the zwittergent series, etc.
  • KCl (10 niM to 2 M); NaCl (10 mM to 4 M); sodium sulfate ( 10 mM to 1.5 M); urea (0.1 M to 8 M); and/or guanidine HCl (0.1 M to 6 M).
  • the buffer may be mixed either with the insect or with comminuted insect parts at a variety of ratios.
  • the ratio may depend upon the number of runs that are carried out. For example, if multiple runs are carried out, the ratio may be skewed to more insects.
  • An example of a ratio that maybe utilized is 1:3 insects to buffer.
  • the insect/insect pieces and buffer may be mixed.
  • the mixing may be carried out in a variety of ways with a number of different apparatuses.
  • the mixed together may be carried out with end over end mixing or with an overhead lightening mixer.
  • the protein(s) of interest may be extracted from the larvae or larval pieces and the pieces are separated from the buffer, which contains the protein(s) of interest. This is illustrated as step 3 in the flowchart shown in Fig. 1. Extraction and clarification can take place either separately or together.
  • the extraction and the separation may be carried out in a number of different ways. For example, decanting, sieving, screening, low speed centrifugation, counter current extraction, decanter centrifugation, hollow tube or large bore hollow fiber or plate and frame tangential flow filtration, and/or percolation extraction could be utilized. More than one extraction and/or separation step may be carried out. Also, one or more different processes may be utilized in the extraction and separation.
  • Both the extraction and the separation technique(s) utilized may depend at least in part upon the protein(s) involved and the size of the insect pieces, among other factors.
  • the extraction may take about 15 to about 45 minutes.
  • the time range for extraction of the protein or material of interest depends largely on the protein or material of interest and on the buffers used in the extraction process and can range from 15 seconds to up to 4 hours.
  • Some embodiments carry out extraction for about 30 seconds to about 1 minute, about 5 minutes to about 10 minutes, or about 1 hour to about 2 hours.
  • Some specific embodiments utilize 5, 10, 15, 20, 30, 45 or 60 minute periods that the buffer and larvae and/or pieces were mixed. The larvae and/or pieces may be mixed with the buffer once or multiple times and the resulting buffer combined.
  • extraction parameters can include temperature, centrifugation speed and nominal molecular weight cut-off (NMWCO) range for potential tangential flow filtration steps.
  • NMWCO nominal molecular weight cut-off
  • extraction may be any combination of
  • Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation for extraction may be carried out at a temperature of about 4° C or about 20° C. Centrifugation
  • the nominal molecular weight cut-off (NMWCO) range for potential tangential flow filtration steps for extraction may be about 100 kDa NMWCO to about 0.22 microns.
  • Clarification may be carried out at a temperature range of about 4° C to about 45° C.
  • clarification was carried out at a temperature of
  • Centrifugation that may be carried out for clarification may be carried out at a
  • clarification may be carried out with a screen size of about 0.01 mm to about 1 mm.
  • One particular embodiment was carried out with a screen size of about 0.5 mm.
  • the nominal molecular weight cut-off (NMWCO) for potential tangential flow filtration steps for clarification may range from about 100 kDa NMWCO to about 0.22 microns.
  • the protein(s) may be isolated from the buffer. This may be carried out utilizing known methods for isolating proteins. For example, proteins may be isolated by any combination of tangential flow filtration, liquid-liquid extraction, column chromatography, precipitation, membrane binding, and/or any other known process.
  • the isolation process(es) may be carried out until achieving a desired degree of purity of the protein.
  • the protein is processed until reaching at least about 85% purity. More typically, the protein is at least about 90% pure. In some cases, the protein is at least about 95% pure.
  • the protein may be processed until it has degree of purity that is typically necessary for the protein to have effectiveness for its end use.
  • the invention may be utilized to isolate any protein product or other desired materials or components from the organism involved.
  • any protein that is or could be expressed by insect larvae, whether native or recombinant could be isolated.
  • Examples include LlR, B5R, A33R, all being truncated, secreted forms of three viral proteins.
  • Other examples include Fab (Fab fragment of a monoclonal antibody, a secreted protein), and DsRed (a non-secreted fluorescent protein composed of a homotetramer).
  • Another example may be a monoclonal antibody (Mab).
  • Fig. 2 illustrates an embodiment of a system according to the present invention. This embodiment may be utilized to carry out the method described above.
  • This embodiment of the system receives whole larvae, pieces of larvae, whole insects and/or pieces of insects on a screw conveyor 5.
  • the screw conveyor transports the larvae to a mill 7 for chopping the larvae. After passing through the mill, the chopped larvae are transported by screw conveyor 9 to mixing vessel 11.
  • Extraction buffer stored in a reservoir 13 is pumped by pump 15 to mixing vessel 11.
  • the chopped larvae and extraction buffer are mixed in the mixing vessel.
  • the chopped larvae and extraction buffer are sent to a separator 17 to separate extract containing protein from larval debris.
  • a final extraction buffer stored in reservoir 19 may be pumped by pump 21 into the separator 17. After separation, the buffer is transported to a receiving vessel 23 and larval debris is sent to receiving vessel 25.
  • the present invention permits the non-manual isolation of proteins contained in the hemolymph of insect larvae.
  • the separation may be carried out while leaving remaining, non-desired larval debris behind in a scalable fashion.
  • Gut proteases may be separated from desired larval material in the case of some non-secreted proteins and in the case of membrane bound or membrane associated proteins.
  • Larvae were utilized expressing truncated LlR, which is a secreted viral protein.
  • the larvae were frozen. Either whole larvae or larvae fractured by impacting with a hammer or mallet were mixed end over end at room temperature in 50 ml conical tubes with an extraction buffer.
  • the extraction buffer in this case was 5OmM Tris, pH 7.8 with 150 mM NaCl and 5 mM beta-mercaptoethanol.
  • the extracted hemolymph containing the LlR was separated from the remaining larval debris by decanting the extraction buffer from the 50 ml conical tubes, leaving the larval debris behind. Analysis performed by Western blot yielded the results shown in Fig. 3 and Table 1 below, which provides densitometry values.
  • LlR was detected with a rabbit polyclonal specific for LlR. Levels were determined by densitometry using a Kodak 1 imaging system. Protein levels were determined by Coomassie Plus assay with BSA as the standard. The three lanes shown in Fig. 3 are 1) cracked larvae, 2) whole larvae, and 3) ground larvae. The level of LlR extracted from the fractured larvae was similar to the level observed from totally homogenized larvae. However, the extracted protein included fewer larval contaminants and required far less work to clarify the material for further processing.
  • Larvae were utilized expressing truncated LlR, B5R and A33R, which are all truncated secreted forms of three viral antigens, and a FAB.
  • the larvae were frozen. Either whole larvae or larvae fractured by impacting with a hammer or mallet were mixed end over end at room temperature in 50 ml conical tubes with an extraction buffer.
  • the extraction buffer in this case was 5OmM Tris, pH 7.8 with 150 mM NaCl and 5 mM beta- mercaptoethanol.
  • the extracted hemolymph containing the LlR was separated from the remaining larval debris by decanting the extraction buffer from the 50 ml conical tubes, leaving the larval debris behind.
  • Fig. 4 illustrates that the ability to extract proteins from larvae is protein independent.
  • DsRed which represents results of extraction of DsRed from whole or partially cracked larvae
  • the partial extraction of DsRed is still occurring after minutes, with the extraction of the larvae chopped at 9000 rpm substantially better then at the lower speeds, probably due to the smaller size of the larval particles.
  • the DsRed was detected using either a semi-qualitative SDS-PAGE Coomassie stained gel or with a western blot with a DsRed specific polyclonal antibody. Densitometry on the bands was performed using the Kodak 1 imaging system.
  • Larvae infected with a baculovirus expressing a humanized MAb were utilized. The larvae were frozen larvae and broken by impacting between two plates. 50 g of larvae were mixed for twenty minutes in a vessel with 400 ml of extraction buffer using an overhead lightening stirrer, which is scalable up to hundreds of liters using large stainless steel vessels with impellers. The larval debris were separated from the extraction buffer using decantation and sieving and analyzed by Western Blot. The protein was extracted with approximately 90% of the MAb being extracted during the first round of mixing. Table 2 below also illustrates results of Example 5.
  • Frozen larvae expressing either LlR (a secreted protein ) or DsRed (a non-secreted protein) were fractured using a milling apparatus, such as the Comil available from Quadro, Inc.
  • the fracturing was carried out under a range of conditions including variations in screen pore size, cutting surface, speed, and temperature.
  • Screen port size varied from about 2 mm to about 9 mm.
  • Various cutting surface configurations included square, round, aspect, flat, and raised.
  • the speed was varied from about 3200 rpm to about 5000 rpm.
  • the temperature of the milling ranged from about 25°C to about -196°C. In this example, the impeller speed was about 3500 and the impactation surfaces were cooled with liquid nitrogen.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Insects & Arthropods (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé pour récupérer des protéines à partir de larves d'insectes. Selon cette invention, un tampon d'extraction est mélangé à des larves d'insectes entières et/ou à des parties non homogénéisées de larves d'insectes, le tampon est ensuite séparé des larves d'insectes entières et/ou des parties non homogénéisées de larves d'insectes, puis les protéines sont isolées du tampon séparé.
EP05821398A 2004-11-12 2005-11-14 Procede pour recuperer des proteines a partir de larves d'insectes Withdrawn EP1824974A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US62708904P 2004-11-12 2004-11-12
PCT/US2005/041038 WO2006053253A2 (fr) 2004-11-12 2005-11-14 Procede pour recuperer des proteines a partir de larves d'insectes

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EP1824974A2 true EP1824974A2 (fr) 2007-08-29

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EP (1) EP1824974A2 (fr)
JP (1) JP2008519855A (fr)
CA (1) CA2587547A1 (fr)
WO (1) WO2006053253A2 (fr)

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KR102652252B1 (ko) * 2018-05-29 2024-04-01 한국생산기술연구원 식물체 유래 단백질의 정제 및 추출 시스템
EP3578052A1 (fr) * 2018-06-05 2019-12-11 Bühler Insect Technology Solutions AG Traitement de larves d'insectes

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JPH0919238A (ja) * 1995-07-05 1997-01-21 Toray Ind Inc 蚕の切開方法
EP0927515B1 (fr) * 1997-04-28 2000-09-20 JAPAN as repr. by DIR. GENERAL of NATIONAL INST. OF SERICULTURAL & ENTOMOLOGICAL SCIENCE MINISTRY OF AGR, FORESTRY & FISHERI Procede de collecte d'humeurs d'insectes
JP2002034590A (ja) * 2000-07-28 2002-02-05 Toray Ind Inc 有用タンパク質の製造方法、イヌ疾病の治療剤およびイヌ疾病の治療方法

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WO2006053253A3 (fr) 2006-08-03
JP2008519855A (ja) 2008-06-12
WO2006053253A2 (fr) 2006-05-18

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