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Effect of silkworm hemolymph on N‐linked glycosylation in two Trichoplusia ni insect cell lines
Author(s) -
Joosten Christoph E.,
Park Tai Hyun,
Shuler Michael L.
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10696
Subject(s) - hemolymph , glycosylation , biology , mannose , cell culture , biochemistry , trichoplusia , glycoprotein , recombinant dna , microbiology and biotechnology , fetal bovine serum , cell , lepidoptera genitalia , botany , gene , genetics , noctuidae
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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 695–705, 2003.