Modifying Secretion and Post-Translational Processing in Insect Cells Eric Ailor and Michael J Betenbaugh*

142 Modifying secretion and post-translational processing in insect cells Eric Ailor and Michael J Betenbaugh* The baculovirus–insect cell system is a valuable tool for the An innovative approach to overcome these limitations in expression of heterologous proteins. Due to limitations in the the baculovirus–insect cell system is to engineer the intracellular processing environment, however, heterologous secretory pathway by supplementing secretory process- secreted and membrane proteins are often insoluble, poorly ing proteins absent or limited in supply in the host insect processed, or contain ‘non-human’ modifications. Recent cell intracellular environment. The focus of this paper is attempts to modify the insect cell secretory pathway by to review secretory processing in insect cells and high- overexpressing processing factors have demonstrated the light recent attempts to improve secretion and potential to overcome these limitations. post-translational modifications in insect cells by overexpressing heterologous proteins involved in these Addresses processing steps. Department of Chemical Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA The secretory pathway *e-mail: [email protected] The eukaryotic secretory pathway is a complex multi- Current Opinion in Biotechnology 1999, 10:142–145 organelle system which provides for the folding, assembly, post-translational modification, and transport of newly http://biomednet.com/elecref/0958166901000142 translated polypeptides. Associated with each of these © Elsevier Science Ltd ISSN 0958-1669 compartments is a collection of cellular proteins which Abbreviations facilitate the secretion process. BEVS baculovirus expression vector system BiP immunoglobulin heavy chain binding protein Cytosolic processing and chaperones ER endoplasmic reticulum Secreted proteins are initially translated from ribosome- Glc glucose bound mRNA in the cytosol prior to translocation across GlcNAc N-acetylglucosamine IgG immunoglobulin G the endoplasmic reticulum (ER) membrane. Polypeptide Man mannose translocation across the ER membrane can occur either PDI protein disulfide isomerase by a cotranslational or post-translational mechanism [7]. SP signal peptidase Transport in mammalian cells is primarily cotranslational, TGF transforming growth factor though both post-translational and cotranslational routes are used in yeast. The predominant mechanism in insect Introduction cells is not currently known. The baculovirus expression vector system (BEVS) is one of the major recombinant DNA expression systems used The cytosolic chaperone hsp70 is believed to assist in today for the production of a wide variety of heterologous the translocation process by maintaining cytosolic proteins [1•]. One of the major advantages of BEVS is polypeptides in a translocation-competent form. In addi- that it can be used to produce relatively large quantities tion, this cytosolic chaperone may suppress aggregation of post-translationally modified heterologous proteins of these polypeptides by preventing nonspecific inter- [2•]. These proteins are used in a number of applications, molecular hydrophobic interactions [8]. including diagnostics, structure-function studies, and vaccines. A large fraction of the recombinant protein pro- In coexpression studies of human hsp70 and murine duced in insect cells using BEVS, however, can immunoglobulin G (IgG) in insect cells using BEVS, sometimes be poorly processed and accumulate as aggre- hsp70 was shown to bind to IgG chains in soluble cell gates [3••]. The production of very high levels of fractions and enhance solubility of the IgG light chain polypeptide with the strong baculovirus polyhedrin pro- precursor [9••]. As a result of higher soluble levels of the moter and the shut down of host protein synthesis light chain precursor, subsequent processing of the light following baculovirus infection may lead to a limitation in chain precursor into its mature form led to an increase in the supply of secretory assistance factors [4,5]. In addi- secreted levels of IgG. tion, although post-translational processing in insect cells is more similar to mammalian cells than bacteria and Eukaryotic cytosolic hsp70 functions in concert with a col- yeast, it is not always identical and, for applications such lection of cofactors, including hsp40 [10], Hip [11], and as therapeutic proteins, this may be critical [6]. Improper Hop [12], in a cycle of binding and release of target secretory processing can be especially problematic at sev- polypeptides which enhances the chaperoning efficiency eral days postinfection when the host cell’s of hsp70. Coexpression of these cofactors may, therefore, post-translational processing machinery has deteriorated. be necessary to optimize hsp70 function. Modifying secretion and post-translational processing in insect cells Ailor and Betenbaugh 143 ER processing Like cytosolic hsp70, BiP functions optimally in the Following translocation, the polypeptides fold, assem- presence of cofactors in a similar binding and release ble, and may undergo additional processing in the ER. cycle. Modeling studies have indicated that BiP’s activi- As this environment includes very high concentrations of ty is not yet optimized when the chaperone is expressed unfolded polypeptides, chaperones, foldases, and other independently [22•]. Altering the kinetics of BiP release enzymes are necessary to assist folding and limit aggre- from a target polypeptide by coexpressing a cofactor gation of the polypeptides [13]. could reduce polypeptide aggregation and further increase secreted protein yields. Signal peptide processing For secreted and many membrane-associated proteins, a There are a number of additional chaperones, including signal peptide at the amino-terminus is responsible for calnexin and calreticulin, within the ER compartment targeting the polypeptide to the ER [14]. In some cases, that act independently or in concert with other factors to however, the signal peptide may remain attached to the facilitate processing of both secreted and membrane polypeptide indicating improper processing in the secre- proteins along the correct folding pathway. Calnexin has tory pathway. Tessier et al. [15] noted the accumulation been identified as a membrane-bound chaperone which of prepropapain aggregates in the cytoplasm of infected associates with a number of membrane proteins [23] and insect cells and modified the prepropapain to include a secreted glycoprotein folding intermediates [24] prior to insect-cell-derived signal peptide from bee melittin. their folding and transport from the ER. Calreticulin is The replacement of the heterologous prepropapain sig- the ER lumen homolog of calnexin and exhibits many of nal peptide with the honeybee melittin signal peptide the same binding characteristics as calnexin. Calnexin resulted in a fivefold increase in propapain secretion. appeared to be instrumental in facilitating the assembly The use of alternative signal peptides including bee of class I histocompatability molecule heavy chains in melittin, cecropin B, and baculovirus structural 64K gly- Drosophila melanogaster cells [25]. Recent research indi- coprotein, however, does not ensure the proper cates that overexpression of calnexin and calreticulin processing of recombinant proteins [16]. For example, using BEVS can also assist folding and assembly of aggregates of precursor single chain antibody fragments membrane proteins in Spodoptera frugiperda cells (scFv) using the bee melittin signal peptide can accu- (CG Tate, EM Whiteley and MJ Betenbaugh, unpub- mulate following expression in insect cells [17••]. lished data). Cleavage of the amino-terminal signal peptide in the ER is Whereas chaperones prevent intermediates from follow- accomplished by signal peptidase (SP). SP is a single mem- ing unproductive folding pathways, catalytic enzymes in brane protein in bacteria and a protein complex in the ER can accelerate folding along the proper pathway. eukaryotes; however, both bacterial and eukaryotic SPs In this way, these cellular enzymes collaborate with can cleave signal peptides from either source interchange- chaperones to maximize production of functional pro- ably [18]. A recent study demonstrated that overexpression teins. Disulfide bond formation occurs as polypeptides of a bacterial signal peptidase enhanced the processing of pass into the ER, which is a more oxidizing environment a single chain antibody fragment precursor [17••]. than the cytosol. The ER enzyme protein disulfide isomerase (PDI) is known to catalyze oxidization, ER chaperones and foldases reduction, and isomerization of disulfide bonds in vitro As in the cytosol, chaperones act to improve protein fold- [26]. A recent study has demonstrated that overexpres- ing and assembly in the ER by binding to regions of sion of PDI increases the folding and secretion of nascent polypeptides that would otherwise form improp- heterologous IgG from insect cells [27]; however, a er associations and aggregate. Immunoglobulin heavy mutation in the PDI carboxyl-terminus catalytic site can chain binding protein (BiP) is an ER-resident chaperone actually reduce protein solubility in vivo in an anti-chap- of the hsp70 protein family that complexes with a num- eroning role [28•]. The presence of the mutations may ber of polypeptides destined for secretion [19] and may destabalize the PD1 molecule and cause it to aggregate also be involved in assisting translocation of polypep- with other polypeptides

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