EP1824974A2

EP1824974A2 - Recovery of proteins from insect larvae

Info

Publication number: EP1824974A2
Application number: EP05821398A
Authority: EP
Inventors: Richard W Welch; Susan G Brown; James H Campbell; Stuart E Builder
Original assignee: Chesapeake Perl Inc
Current assignee: Chesapeake Perl Inc
Priority date: 2004-11-12
Filing date: 2005-11-14
Publication date: 2007-08-29
Also published as: JP2008519855A; WO2006053253A2; CA2587547A1; WO2006053253A3

Abstract

A method for recovery of proteins from insect larvae is provided. An extraction buffer is mixed with 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.

Description

RECOVERY OF PROTEINS FROM INSECT LARVAE

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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. Currently, 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. Also, 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.

Alternatively, the insect larvae are completely homogenized. However, 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. Additionally, 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.

In another process, 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.

SUMMARY OF THE INVENTION

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. BRIEF DESCRIPTION OF THE DRAWINGS

Objects and advantages of the present invention will be more clearly understood from the following specification when considered in conjunction with the accompanying drawings, in which:

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; and

Fig. 7 is a graph that shows results of Example 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Embodiments of the present invention provide a method and a system for extracting hemolymph from insects. In particular, 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. For example, 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. Additionally, 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. Having to separate the desired target protein(s) from such other debris created or increased by the complete homogenization increases the difficulty and cost of the process, thereby increasing the desirability of the instant method which is not limited by the difficulties associated with complete homogenization of the larvae (infected or non-infected). While the present invention has been described in connection with insects, it may also be utilized to process other organisms, such as spiders, mites or worms. Examples of insects that could be processed according to the present invention 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), tlirips (Thysanoptera), true bugs, flower bugs (Heteroptera), aphids, planthoppers, leafhoppers, scale insects (Homoptera), Lacewings (Neuroptera), butterflies, moths (Lepidoptera), beetles (Coleoptera), Flies (Diptera), parasitic wasps, and sawflies (Hymenoptera).

Any protein produced utilizing any insect may be recovered. A few examples of 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.

Although whole insects or larvae and/or cut up insects may be utilized, the invention is typically carried out with larvae that are cut or otherwise made not whole. This is indicated in the flowchart shown in Fig. 1, which 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.

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. Typically, 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. According to one particular embodiment, 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.

According to one embodiment, the insect larvae are comminuted with a milling machine. A particular milling machine that may be used is a Fitzmill milling machine. According to one technique, 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. For example, 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. For example, the comminutation may be carried out at a temperature range of

about -100° C to about 30° C. One particular embodiment was carried out at about 4° C.

According to another embodiment, 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. For example, 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. For example, the comminutation may be carried out at a temperature range of

about -196° C to about 30° C. One particular embodiment was carried out at about -190° C. The range of RPM for communitation may depend on the blades used in the comminutation machinery and the orientation of the blades. According to another technique, 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. According to one embodiment, 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. This is step 2 in the flowchart shown in Fig. 1. The extraction buffer may include any components in which the target protein(s) are soluble. According to one embodiment, 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. Other 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. (0.01% to 10%); 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.

Once the insect/insect pieces and buffer are combined, they may be mixed. The mixing may be carried out in a variety of ways with a number of different apparatuses. For example, the mixed together may be carried out with end over end mixing or with an overhead lightening mixer.

After mixing of the insect pieces, 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.

Other extraction parameters that may be controlled can include temperature, centrifugation speed and nominal molecular weight cut-off (NMWCO) range for potential tangential flow filtration steps. Typically, extraction is carried out at a temperature range of

about 4° C to about 45° C. According to two particular embodiments, 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 speed of about 2,000 x g to about 15,000 x g. 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.

According to one particular embodiment, clarification was carried out at a temperature of

about 4° C. Centrifugation that may be carried out for clarification may be carried out at a

speed of about 2,000 x g to 15,000 x g. Additionally, 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.

After separating the protein-containing buffer from other insect portions, 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. Typically, 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. For example, 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. After mixing, 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.

The following represent four working examples that are illustrative of the present invention and are in no way meant to be exhaustive of the parameters that may be utilized to carry out the invention.

Example 1

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.

Table 1 -Results for the extraction of LlR from larvae

Example 2

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. The four target proteins were extracted in varying degrees from the whole larvae in all cases and in all cases, the resulting protein extract was reasonably clear and ready for further processing. A 10% Bis-tris gel was run in MES and transferred to PVDF. Detection was performed using anti-his MAb. Analysis performed by Western blot yielded the results shown in Fig. 4. The fifteen lanes shown in Fig. 4 are as follows: 1) standard; 2-4) LlR at 15, 45, and 60 minutes; 5-7) B5R at 15, 45, and 60 minutes; 8-11) FAB at 15, 30, 45, and 60 minutes; and 12-15) A33R at 15, 30, 45, and 60 minutes. Fig. 4 illustrates that the ability to extract proteins from larvae is protein independent.

Example 3

Both chopped and whole larvae were utilized to compare the results obtained. Whole frozen larvae expressing LlR and DsRed were chopped in a Fitzmill comminuting device, blade side forward, with the sieving screen removed. The larvae were chopped at three different speeds: 3000 rpm, 6000 rpm, and 9000 rpm. Chopped larvae were stored frozen until extraction testing and analysis. Extraction testing was performed using both whole and chopped larvae as described for the small scale laboratory tests using 50 ml conical tubes. The whole or chopped larvae were mixed with the extraction buffer using end over end mixing. Samples were removed over the course of the extraction for analysis to determine both a rough rate of extraction and to determine the overall level of extraction relative to the whole larvae. Analysis was performed using a semi-qualitative Western blot and an LlR specific polyclonal. This analysis produced the results illustrated in Figs. 5 and 6. Densitometry on the bands was performed using the Kodak 1 imaging system. As can be seen in Fig. 5, the extraction of LlR was essentially complete within fifteen minutes with little difference between the larvae chopped at 6000 rpm and those chopped at 9000 rpm. DsRed is a non-secreted protein the presence of which is easily determined due to the protein's characteristic red color. As can be seen in Fig. 6, 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.

Example 4

Both chopped and whole larvae were utilized to compare the results obtained. Whole frozen larvae expressing LlR were chopped in a Fitzmill comminuting device, blade side forward, with the sieving screen removed. The larvae were chopped at about 9000 rpm. Chopped larvae were stored frozen until extraction testing and analysis. Other whole larvae infected with virus expressing LlR were substantially completely homogenized by grinding using a Turrax ultra-homogenizer in extraction buffer, followed by clarification by centrifugation. Extraction testing was performed using both whole and chopped larvae as described for the small scale laboratory tests using 50 ml conical tubes. The whole or chopped larvae were mixed with the extraction buffer using end over end mixing. Samples were removed over the course of the extraction for analysis to determine both a rough rate of extraction and to determine the overall level of extraction relative to the whole larvae. Analysis was performed by an early development phase ELISA for the detection of LlR which produced the results illustrated in Fig 7. The theoretical maximum for the recovery of the LlR from the larvae was determined analysis of the clarified larval extract homogenized via the Turrax ultra-homogenizer. As can be seen in Fig. 7, the extraction of LlR reached the theoretical maximum between about fifteen minutes and about 45 minutes while the extraction of LlR from the whole larvae had not reached the theoretical maximum after about 1 hour. Assuming linearity of extraction the extraction form the whole larvae would have been complete between about 90 and about 120 minutes.

Example 5

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.

Table 2

Example 6

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. Cracked larvae were stored on dry ice after cracking. In this example, larvae cracked under various conditions were extracted for 30 minutes into buffer (50 mM Tris/1 M sodium chloride, 15 mM imidazole, pH 7.7) at about 2-8°C. Target protein extraction was determined using western blot. Total protein extracted was determined using the Coomassie Plus Bradford Assay. Table 3 presents results from various trials of this example. The values are presented as level of protein extracted relative to protein extracted using a total homogenization method. In this example, the larvae were homogenized using a Ultra Turrax T-25 shear homogenizer in buffer.

Table 3

Claims

Claims:We claim:

1. A method for recovery of proteins from insect larvae, the method comprising: mixing with an extraction buffer at least one of whole insect larvae and non- homogenized parts of insect larvae; separating the buffer from the at least one of whole insect larvae and non- homogenized parts of insect larvae; and isolating the proteins from the separated buffer.

2. The method according to claim 1, wherein insect parts are mixed with the extraction buffer, and wherein the insect parts have a length of about 1 mm to about 1 cm.

3. The method according to claim 1, further comprising: fracturing or cutting frozen insect larvae into pieces having a length of about 1 mm to about 1 cm.

4. The method according to claim 1, wherein the buffer is separated from the at least one of whole insect larvae and non-homogenized parts of insect larvae by at least one of 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, or percolation extraction.

5. The method according to claim 1, wherein the proteins are native.

6. The method according to claim 1, wherein the proteins are recombinant.

7. The method according to claim 1, wherein the extraction buffer and the at least one of whole insect larvae and non-homogenized parts of insect larvae are mixed together with end over end mixing.

8. The method according to claim 1, wherein the extraction buffer and the at least one of whole insect larvae and non-homogenized parts of insect larvae are mixed together with an overhead lightening mixer.

9. The method according to claim 1, further comprising: fracturing the larvae prior to mixing with the extraction buffer.

10. The method according to claim 9, wherein the larvae are fractured by impacting with a hammer or mallet.

11. The method according to claim 9, further comprising: freezing the larvae prior to fracturing.

12. The method according to claim 1, wherein the extraction buffer comprises 50 mM Tris having a pH of 7.8, 150 mM NaCl, and 5 mM beta-mercaptoethanol.

13. The method according to claim 1, wherein the insect larvae are raised in a web of wells, with each well containing at least one larva and diet, the method further comprising: chopping up the web of wells, diet and larvae.

14. The method according to claim 1, wherein the proteins are recovered from hemolymph of the larvae.

15. The method according to claim 1, wherein the insect larvae are Trichophisia nϊ larvae.

16. The method according to claim 1, wherein the proteins include at least one of 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 interleukin-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.

17. The method according to claim 1, wherein hemolymph of the larvae is isolated from the at least one of whole insect larvae and non-homogenized parts of insect larvae and the proteins are isolated from the hemolymph.

18. The method according to claim 1, wherein the extraction buffer and the at least one of whole insect larvae and non-homogenized parts of insect larvae are mixed together for a period of about 15 minutes to about 45 minutes.

19. The method according to claim 1, wherein the extraction buffer and the at least one of whole insect larvae and non-homogenized parts of insect larvae are mixed together for a period of about 15 seconds to up to 4 hours.

20. The method according to claim 1, wherein the extraction buffer and the at least one of whole insect larvae and non-homogenized parts of insect larvae are mixed together for a period of about 30 seconds to about 1 minute.

21. The method according to claim 1, wherein the extraction buffer and the at least one of whole insect larvae and non-homogenized parts of insect larvae are mixed together for a period of about 5 minutes to about 10 minutes.

22. The method according to claim 1, wherein the extraction buffer and the at least one of whole insect larvae and non-homogenized parts of insect larvae are mixed together for a period of about 1 hour to about 2 hours.

23. The method according to claim 1, further comprising: purifying the isolated proteins.

24. A system for recovery of proteins from insect larvae, the system comprising: a comminuting device to cut up the larvae into pieces or press the larvae into flakes; a mixing vessel operative to receive the larvae pieces or flakes and an extraction buffer; a container operative to receive extraction buffer after mixing with the larvae pieces or flakes; and a separator operative to separate the proteins from larval debris.

25. The system according to claim 24, further comprising: a freezer operative to freeze the larvae; and fracturing apparatus operative to fracture frozen larvae.

26. A system for recovery of proteins from insect larvae, the system comprising: means for producing pieces or flakes of larvae; means for combining the larvae pieces or flakes and an extraction buffer such that larval proteins are extracted into the buffer; and means for isolating larval proteins from the extraction buffer.

27. The system according to claim 26, further comprising: freezing means for freezing the larvae; and fracturing means for fracturing frozen larvae.