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Baculovirus vectors for expression in insect cells lan Jones* and Yuko Morikawat

Recombinant baculoviruses now represent a mature technology in which vector development, particularly for the control of expression level, has reached a plateau. However, other aspects of expression, such as the production of multiple proteins, improved product purification or maximizing protein processing, remain areas for novel vector and host cell development. This year has seen these topics come to the fore in descriptions of new expression systems.

Addresses * Institute of Virology, Mansfield Road, Oxford OXl 3SR, UK; e-mail: ijones@molbiol.ox.ac.u k tThe Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108, Japan

Current Opinion in Biotechnology 1996, 7:512-516

@ Current Biology Ltd ISSN 0958-1669

Abbreviations AcMNPV Autographa califomica multiple nuclear polyhedrosis virus Ea Estigmene acrea GST glutathione S-transferase LIC ligation-independent cloning PCR polymerase chain reaction VLP virus-like particle

I n t r o d u c t i o n It is now more than 10 years since the first manipulation of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) for the expression of non-baculoviral proteins was demonstrated [1]. In the intervening years, the basic principle of recombinant formation, integration of a foreign gene into the viral genome at a site of high transcriptional activity has changed little, but the understanding of the process and its directed manipulation have progressed dramatically. Currently, with the -135 kilobase (kb) sequence of the viral genome determined [2], it is possible to insert any gene at any genomic location. The number of loci where insertion leaves the virus viable, however, remains subject to experimental test. Although the techniques involved are now commonplace (for a recent review, see [3]), a brief revision of the process of recombinant formation is appropriate here to highlight the areas that would benefit from improvement.

In principle, there are two methods of recombinant formation differing only in the location at which the gene of interest is incorporated into the baculovirus genome. In still the most common method of recombi- nation, a transfer vector containing the cloned gene is introduced, with viral DNA, into insect cells where the recombination event takes place. The use of wild-type unmodified viral DNA for this process has now been largely replaced by the use of linear forms of the viral

genome unable to initiate an infection unless rescued to the circular replication-competent form by recombination [4]. This process is efficient but relies on two factors: the construction of a transfer vector in which the gene of interest is positioned under the control of a strong baculoviral promoter and flanked by baculovirus DNA for recombination, and the use of high-quality linear viral DNA with the minimum possibility of recircularization via non-desired recombination events. For the polyhedrin locus, by far still the most exploited transcription signals for the construction of recombinant baculoviruses, these two criteria are easily satisfied: a variety of vectors exist that contain multiple sites for insert cloning and viral DNA has been engineered such that linearization via enzyme sites in the polyhedrin locus give essentially no background in the absence of recombination with the transfer vector. These criteria are not as well catered for, however, if the desired aim is to produce a recombinant with a nonpolyhedrin promoter or at an alternate locus in the genome.

In the second method of recombinant baculovirus forma- tion, the introduction of genetic material into the viral genome occurs outside of the insect cell and modified recombinant baculoviral DNA is the only DNA introduced into insect cell culture. There are three flavours of the latter technology: recombination in Escherichia coli [5], recombination in yeast [6], and recombination in vitro [7]. In principle, these technologies offer greater flexibility and, because recombinant viral DNA is characterized prior to its introduction into cells, there is formally no need to further isolate a single recombinant virus by plaque assay. However, the ex vivo methods for recombinant formation are recent and their development currently lags behind in vivo recombination despite their technical superiority. The further development of recombination methods is undoubtedly one of the areas of future growth and improvements within the last year are described below.

Vector design in the early years of recombinant bac- uloviruses was almost wholly directed at maximizing the level of protein expressed and almost all current vectors based on the polyhedrin locus now contain the essential elements necessary for this, most importantly, the full length nontranslated polyhedrin leader sequence prior to the ATG start codon of the inserted sequence. It is important to realise however, that maximal rates of transcription and translation may not guarantee maximum yields of a final protein if that protein requires extensive post-translational modification such as protein cleavage or glycosylation. The past year has seen further attempts to improve the production of such 'difficult' proteins by the description of vectors and cell lines designed

BacuIovirus vectors for expression in insect cells Jones and Morikawa 513

to modify the expressed protein. The purification of proteins from a recombinant source may also be a limiting factor in obtaining sufficient protein for desired structure and function studies. Thus, the inclusion of tags to enable purification of recombinant forms of protein is an increasing trend that has been further demonstrated in the last year.

N e w m e t h o d s of r e c o m b i n a t i o n Two developments have occurred in the past year and both represent improvements in existing technologies. In the first, the commercial release of a set of baculovirus vectors from Novagen Inc. has included a recombination system based on ligation-independent cloning (LIC) [8"]. In this system, the gene of interest is amplified using the polymerase chain reaction (PCR) and primers with specified oligonucleotide extensions that lack thymine residues. The PCR product is treated with T4 DNA poly- merase in the presence of excess dATP to produce long single-stranded overhangs that anneal to complementary tails in the vector pBAC LIC and the mixture is used directly with cut viral DNA to transfect insect cells. The avoidance of a cloning step in E. coli speeds up the process of recombinant formation but the lack of any verification of the ligation product prior to transfection theoretically leaves the system open to a higher than usual background as a result of recombination via aberrant ligation products.

In a second system described within the past year, improvements have been made to the methods for insertion into the baculovirus genome whilst it is held as a large plasmid, a bacmid, in E. coli, as first reported by Luckow etal. [5]. The original system suffered from a poor selection procedure for the transposition events that led to the insertion of the gene of interest into the baculovirus genome. Now, by the use of a temperature-sensitive se- lection methodology combined with a blue/white selection phenotype, these problems have been overcome [9"]. Efficiencies of up to 105 recombinants per microgram of DNA were possible, making the concept of baculovirus cDNA libraries for direct screening of products viable. Library formation in baculoviruses represents one of the current limitations for a number of technologies (see section 'New directions' below) and either of the developments in recombination described here may improve the efficiencies that are currently possible. Note that both of the improvements in recombination described have been designed for the polyhedrin locus only.

Express ion vectors Single promoter vectors As described in the introduction, the need to ensure high-level synthesis of protein has largely been satisfied in the vectors now in common use. Recent vectors based on a single promoter have thus concentrated on ensuring that the expressed protein is modified in an appropriate manner or can be purified simply. A case in point is protein glycosylation, which relies on efficient transport

of the recombinant protein through the endoplasmic reticulum and the action of a number of glycosidases and glycotransferases. Neither process, it is argued, operates efficiently in the infected cell late in the viral life cycle when the polyhedrin promoter is most active. To overcome this, Chazenbalk and Rapoport [10"] report the construction of a vector (pAcMP3) with a late basic protein promoter, which is active earlier in the infectious cycle than is the polyhedrin promoter. The level of glycosylation of a test protein, thyrotropin (TSH) receptor, was improved by the use of this vector when compared to the polyhedrin-based pVL1393, as was the level of bioactivity exhibited by the final protein. The overall yield of protein was, however, not improved and the general usefulness of this type of vector may depend on the nature of the glycoprotein expressed. Glycoproteins whose activity does not depend on the nature or extent of carbohydrate added will continue to be best expressed by the stronger polyhedrin promoter whilst those where glycosylation is a key regulator of activity (see, for example, [11,12"]) may be improved by this strategy. Note that glycoprotein folding, glycosylation and secretion are closely linked and examples of other methods to improve one or all of these are included in the 'Host cells' section below.

Poor signal peptide recognition and cleavage is another cause of poor glycoprotein expression and, in the case of proteins that lack transmembrane domains, secre- tion. Vectors designed to maximize secretion have been reported by Wang et al. [13] and Kuhn and Zipfel [14"]. In both cases, the vector encodes a well-processed signal peptide and is combined with an affinity tag for protein purification following secretion. In the former, the baculovirus major glycoprotein gp67 signal sequence was used with glutathione S-transferase (GST) fused in frame to provide an affinity domain 5' to the inserted coding region. In the latter example, the signal peptide from human factor H-like protein 1 was used with an eight amino acid histidine tag positioned 3' to the cloning site to provide a carboxy-terminal poly-histidine extension to enable metal chelate chromatography. In both cases, the vectors are driven by the polyhedrin promoter and the affinity tags could be removed by site-specific proteolysis. Histidine tags are a popular way of enabling recombinant protein purification (see, for example, [15]) but in two reports this year, the histidine tag itself has been shown to be involved in the activity of the final purified recombinant protein [16",17"]. There is a continuing need, therefore, to explore the use of other protein purification tags for inclusion in future baculovirus vectors.

There is constant low-level interest in the development of vectors based on strong promoters other than the polyhedrin, either to allow insertion at separate loci within the genome or to try to bypass patents that specify the use of the polyhedrin promoter. The pl0 promoter has been suggested for this purpose and a system for the generation

514 Expression systems

of pl0-based recombinants has been described within the year [18].

Multiple promoter vectors The production of two or more proteins at high level within the same cell allows interactions between them to occur readily. Vectors have been developed to allow the expression of two, three or four proteins within the same infected cell, as shown by Belyaev etal. [19]. These authors also show that co-infection of two recombinant viruses is efficient, resulting in the possibility of producing eight proteins within a single infected cell at the same time. This might allow the construction of complex biochemical or assembly pathways, in which each of the participating proteins could be manipulated at ease whilst the levels of all others remained constant. The use of these vectors for the assembly of a complex multilayered viral structure has recently been reviewed [20].

Vectors for specific uses Whilst it may be generally accepted that current vectors ensure that levels of expressed protein are near their maximum, the development of novel vectors for specific uses continues to advance. Poul et al. [21] have reported baculovirus vectors for the production of recombinant antibodies in insect cells. This system produces whole rather than single-chain antibodies by the co-expression of light and heavy chains in the same cell. The vectors contain the antibody constant regions and cassette cloning sites for any light and heavy variable domains under the control of the polyhedrin and pl0 promoters.

The use of virus-like particles (VLPs), produced by recom- binant baculoviruses, for carriage of epitopes suggested as useful for retrovirus immunization has been reported by Brand et al, [22]. A region of coding sequence of the gag gene of HIV-1 known not to be required for VLP assembly and to represent a known epitope is replaced by a sequence from the HIV envelope gene. Following expression, VLPs continue to assemble and now present envelope epitopes in the context of a particulate structure. Similar in principle, the use of native VLPs to pick up foreign envelope proteins from the surface of infected cells has also been reported [23].

Host cells for protein expression Historically, the concentration of effort in the development of expression vectors and of improved ways of generating and selecting recombinants has left unexplored the modification of the host cell as a method of altering the nature of the expressed protein. This trend is now changing. Two years ago, Hsu et al. [24] reported the co-expression of a chaperonin molecule, BiP, with antibody chains to improve the level of folded recombinant protein produced. Results were positive, though not dramatic, but the point of engineering the pathway of expression rather than solely the level of expression was well made. Gross changes in the pathway of expression may be made by

changing the cell line used for protein expression. This year, two reports [25°,26 °] have confirmed that the use of a different cell line to the Spodoptera frugipefda cells commonly used for expression can dramatically alter the pattern of glycosylation of the recombinant glycoprotein produced. The original observations that Estigmene acrea (Ea) cells produce a glycosylation pattern more closely akin to mammalian than insect cells was made some years ago [27] but further examination of this cell line has only been published this year. A clonal line, Ea4, derived from the original Ea culture, has been reported by Ogonah eta/. [25 °] and the glycosylation pattern has been characterized and confirmed as high in complex sugar. At the same time, Wagner et al. [26 °] have identified the key enzyme in Ea cells that is responsible for the altered glycosylation pattern. As cited above, there are occasions when the high-mannose non-complex sugar additions typical of Spodoptera frugiperda cells do not allow full glycoprotein bioactivity yet where the yield of protein common in the baculovirus system is desirable. In such cases, the use of Ea cells for the production of mammalian-like glycosylation patterns may be very valuable.

New directions Two developments in the past year merit inclusion as new directions for the exploitation of baculoviruses in the future. Boublik et al. [28] have reported the use of AcMNPV as a vector for the display of proteins on the virus surface. This development relied on the insertion of foreign proteins into the major baculovirus surface glycoprotein gp67 at a site between the signal peptide and the mature protein without loss of the ability to be transported to the plasma membrane for virion incorporation. The technology holds promise for the forced in vitro evolution of complex glycoproteins in the same way as phage display is applied to antibody fragments. Current limitations to the technology, however, mean it would benefit from the improved methods of recombinant selection reviewed above.

A second new direction for baculoviruses with wide-rang- ing and diverse applications is the novel finding that human hepatocytes can efficiently take up baculoviruses and are capable of expressing foreign antigens [29°]. Other cell lines examined did not take up baculovirus as well, suggesting that baculoviruses could be used to specifically target liver cells for gene therapy applications.

Conclusions The past year might justly be considered to have consisted of more consolidation than innovation with respect to the major use of recombinant baculoviruses, that is, the expression of a single protein at a high level. Nevertheless, long-standing protocols continue to be improved such that recombinant viruses can be produced easily and at high frequency and there are a wide variety of vectors available for the expression of a single gene product in nonsecreted or secreted form, with or without affinity tags

Baculovirus vectors for expression in insect cells Jones and Morikawa 515

to aid protein purification. The use of Spodoptera cells with engineered secretion pathways or wholly different cell lines for the expression of glycosylated or secreted proteins is an interesting development that is sure to see further progress in the future. Some findings, such as the use of baculoviruses for the development of display libraries or for gene delivery in non-insect cell types, represent completely new areas that suggest a future for baculoviruses beyond their historical use as workhorses for protein expression.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest • • of outstanding interest

1. Smith GE, Summers MD, Frazer M J: Production of human beta- interferon in insect cells infected with a baculovirus expression vector. Mol Cell Bio/1983, 3:2156-2165.

2. Ayres MD, Howard SC, Kuzio J, Lopez-Ferber M, Posses RD: The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 1994, 202:586-605.

3. Davies AH: Current methods for manipulating baculoviruses. Nat Biotechnol 1994, 12:47-50.

4. Kitts PA, Possee RD: A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 1993, 14:810-817.

5. Luckow VA, Lee SC, Barry GF, Olins PO: Efficient generation of infectious recombinant baculoviruses by site-specific transposon-medisted insertion of foreign genes into a baculovirus genome propagated in Escherichia coil J Viro/ 1993, 67:4566-4579.

6. Patel G, Nasmyth K, Jones N: A new method for the isolation of recombinant baculovirus. Nucleic Acids Res 1992, 20:97-104.

7. Ernst WJ, Grabherr RM, Katinger HW: Direct cloning into the Autographs californica nuclear polyhedrosis virus for generation of recombinant baculoviruses. Nucleic Acids Res 1994, 22:2855-2856.

8. Bishop DHL, Nov,/R, Mierandorf R: The BacVector system: • simplified cloning and protein expression using novel

baculovirus vectors. Innovations 1995, 4:1-6. Application of LIC to the generation of recombinant baculoviruses. The E. coil cloning step normally required for the generation of the transfer vector is avoided and the process is correspondingly much faster.

9. Leusch MS, Lee SC, Olins PO: A novel host-vector system for • direct selection of recombinant baculoviruses (bacmids) in

Escherichia coll. Gene 1995, 160:191-194. An improved method for the generation of recombinant baculoviruses by transposon-mediated recombination in E. coil.

10. Chazenbalk GD, Rapoport B: Expression of the extracellular • domain of the thyrotropin receptor in the baculovirus

system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of functional highly glycosylated proteins. J Bio/Chem 1995, 270:1543-1549.

Interesting in concept, this paper describes the use of an 'early' baculovirus promoter for the production of more highly glycosylated glycoproteins. Over- all yields were unchanged but specific activity was increased.

11. Kawamoto S, Uchino S, Hattori S, Hamajima K, Mishina M, Nakajima-lijima S, Okuda K: Expression and characterization of the zeta 1 subunit of the N-methyI-D-aspartate (NMDA) receptor channel in a baculovirus system. Brain Res Me/Brain Res 1995, 30:137-148.

12. Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin • M, Dong C, Weiner A J, Lau JYN, Choo CI-L eta/.: A quantitative

test to estimate neutralising antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Nat/Acad Sci USA 1996, 93:1759-1763.

An interesting direct comparison between the bioactivity of hepatitis C envelope protein produced in mammalian and insect cells. Both preparations were active but the mammalian version much more so than the baculovirus- expressed product, presumably due to the type of glycosylation added.

13. Wang YH, Davies AH, Jones IM: Expression end purification of of glutathione-S-transferase tagged HIV-1 gp120; no evidence for an interaction with CD26. Viro/ogy 1995, 208:142-146.

14. Kuhn S, Zipfel PF: The baculovirus expression vector • pBSV-8His directs secretion of histidine-tagged proteins.

Gene 1995, 162:225-229. An all-in-one vector for the secretion of protein into the cell medium and affinity purification based on metal chelate chromatography.

15. Amarneh B, Simpson ER: Expression of a recombinant derivative of human aromatase P450 in insect cells utilizing the baculovirus vector system. Mol Cell Endocrinol 1995, 109:R1 -R5 .

16. Janssen J J, Bovee-Geurts PH, Merkx M, DeGrip WJ: Histidine • tagging both allows convenient single-step purification of

bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry. J Biol Chem 1995, 2 7 0 : 1 1 2 2 2 - 1 1 2 2 9 .

A cautionary note for those using the histidine tag for protein purification as it contributes to the activity profile of the expressed enzyme.

17. Pekrun K, Petry H, Jentsch KD, Moosmayer D, Hunsmann G, • Luke W: Expression and characterization of the reverse

transcriptase enzyme from type 1 human immunodeficiency virus using different baculoviral vector systems. Eur J Biochem 1995, 234:811-818.

Another cautionary note on the use of histidine tags: removal by digestion with enterokinase resulted in complete loss of bioactivity.

18. Martens JW, Van-Oers MM, Van-de-Bilt BD, Oudshoorn P, Vlak JM: Development of a baculovirus vector that facilitates the generation of p10-based recombinants. J Viro/Methods 1995, 52:15-19.

19. Belyaev AS, Hails RS, Roy P: High-level expression of five foreign genes by a single recombinant baculovirus. Gene 1995, 156:229-233.

20. Roy P: Orbivirus structure and assembly. Virology 1996, 215:1-11.

21. Poul M-A, Cerutti M, Chaabihi H, Tiechioni M, Deramoudt F-X, Bernard A, Devauchelle G, Kaczorek M, LeFranc M-P: Cassette baculovirus vectors for the production of chimeric, humanised, or human antibodies in insect cells. Eur J/mmunol 1995, 25:2005-2009.

22. Brand D, Mallet F, Truong C, Roingeard P, Goudeau A, Barin F: A simple procedure to generate chimeric Pr55gag virus-like particles expressing the principal neutralization domain of human immunodeficiency virus type 1. J Virol Methods 1995, 51:153-168.

23. Gamier L, Ravallec M, Blanchard P, Chaabihi H, Bossy J-P, Devauchelle G, Jestin A: Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 gag particles produced in baculovirus infected cells. J Viro11995, 69:4060-4068.

24. Hsu TA, Eiden J J, Betenbaugh M J: Engineering the assembly pathway of the baculovirus-insect cell expression system. Ann NY Acad Sci 1994, 721:208-217.

25. O9onah OW, Freedman RB, Jenkins N, Patel K, Rooney BC: • Isolation and charecterisetion of an insect cell line able to

perform complex N-linked glycosylation on recombinant proteins. Nat Biotechno11996, 14:197-202.

Derivation of a clonal line of Ea cells with good growth rates and a mammalian-like glycosylation pattern.

26. Wagner R, Geyer H, Geyer R, Klenk H-D: N-acetyl-beta- • glucosidase accounts for differences in glycosylation of

influenza virus hemagglutinin expressed in insect cells from a baculovirus vector. J Viro11996, 70:4103-4109,

More on Ea cells and the enzymatic machinery that gives rise to altered glycosylation patterns.

27. Klenk H-D, Cramer A, Wagner R, Groner A, Kretzschar E: Processing of influenza virus hemagglutinin in insect cells: variations depending on the host and on co-expression with other viral proteins. In Baculovirus and Recombinant Protein

516 Expression systems

28.

Production Processes. Edited by Vlak JM, Schlaeger E-J, Bernard AR. Basle: Editions Roche; 1992:166-174.

Boublik 'it, DiBonito P, Jones IM: Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa calffornica nuclear polyhedmsis virus (AcNPV) for the presentation of foreign proteins on the virus surface. Nat Biotechnol 1995, 13:1079-1084.

29. Hofmann C, Sandig V, Jennings G, Rudolph M, Schlag P, • Strauss M: Efficient gene transfer into human hepatocytes

by baculovirus vectors. Proc Nat/Acad Sci USA 1995, 92:10099-10103.

A development for baculoviruses that could hardly have been forseen. Hu- man liver cells efficiently take up the virus and, when a reporter gene is driven by a suitable promoter, express protein. Opens many new avenues for baculovirus development, not least of which is the nature of the attachment and uptake.