Effect of silkworm hemolymph on N‐linked glycosylation in two Trichoplusia ni insect cell lines

Abstract

A recombinant N-linked glycoprotein, secreted human placental alkaline phosphatase (SEAP), was produced in two Trichoplusia ni insect cell lines using the baculovirus expression vector. Silkworm hemolymph (SH) was added to TNMFH + 10% fetal bovine serum (FBS) medium to a concentration of 2.5% or 5%, and SEAP production and glycosylation in the presence of SH were compared with controls devoid of hemolymph. Growing Tn-4s cells in 5% SH-supplemented medium required progressive adaptation of the cells to SH, and adapted cells had a SEAP specific yield decreased by 2.5-fold compared with control cells not exposed to SH. Although SEAP produced in the control possessed little complex glycosylation (<1%), SEAP produced by SH-adapted cells in the presence of 5% SH possessed 8.7% sialylated structures, as well as unusual, asialylated, agalactosylated structures with a high degree of polymerization (DP). On the basis of enzymatic and mass-spectrometric analyses, we propose that these structures are glucosylated, high-mannose oligosaccharides. SEAP was also produced by Tn-4s cells without adaptation to SH when SH was added just prior to baculovirus infection, but SEAP specific yield was adversely affected (approximately fourfold reduction compared with control devoid of hemolymph), and glycosylation of SEAP produced under these conditions was characterized by large amounts of high-mannose and high-DP structures and an absence of complex structures. Similarly, Tn5B1-4 cells that were not adapted to SH had a SEAP specific yield reduced by approximately fivefold in SH-containing medium; however, these cells were able to produce 13.5% sialylated SEAP in the presence of 2.5% SH, whereas complex structures were not produced in the absence of SH. We propose that SH improves glycosylation either directly or indirectly by decreasing SEAP specific yield.

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N‐glycosylation of human secreted alkaline phosphatase

“…A higher initial concentration of cells could be used to achieve the same amount of recombinant protein. It should be noted that our results are consistent with other expression systems, where supplementation has had beneficial results on glycosylation and slightly detrimental effects on cell growth and/or recombinant protein production (Donaldson et al, 1999;Gawlitzek et al, 2000;Joosten et al, 2003). In addition, the detrimental effects of the supplements used on plant cell growth have been documented and our data are consistent such previous findings (e.g.…”

Protein‐stabilizing and cell‐penetrating properties of α‐helix domain of 30Kc19 protein

“…In previous studies, the silkworm hemolymph (SH) of Bombyx mori was shown to exhibit anti‐apoptotic properties in insect and mammalian cells. Also, apoptosis was inhibited by 30K gene and recombinant protein from SH .…”

Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT‐1 cells cultured in serum‐free medium

“…This limitation reflects a fundamental difference between the N-glycan processing pathways of lepidopteran insect and mammalian cells, as most evidence indicates that the former lack functional levels of sialyltransferases (Butters et al, 1981;Stollar et al, 1976) and CMP-sialic acids (Hooker et al, 1999;Tomiya et al, 2001). Clearly, there are some published results that raise questions about the overall generality of this conclusion as it applies to lepidopteran (Davidson et al, 1990;Joosten et al, 2003;Shuler, 2003a, 2003b;Joshi et al, 2001;Palomares et al, 2003;Watanabe et al, 2001) and other (Kim et al, 2002;Koles et al, 2004) insect systems. However, the fact remains that most recombinant N-glycoproteins produced by the most commonly used baculovirus-insect cell combinations are not terminally sialylated.…”

“…Thus, it is clear that it will be necessary to survey the ability of this transgenic insect cell line to support sialylation of a much larger and broader array of recombinant glycoprotein products before drawing any sweeping conclusions. In context of this discussion, it is interesting to note that many of the studies reporting recombinant glycoprotein sialylation with the baculovirusinsect cell system have used human secreted alkaline phosphatase as the model glycoprotein (Joosten and Shuler, 2003a, b;Joosten et al, 2003Joosten et al, , 2001Palomares et al, 2003). Perhaps this particular product has some biophysical properties that make its N-glycans particularly amenable to elongation or resistant to deglycosylation.…”