ε-N-Acetylation in the Production of Recombinant Human Basic Fibroblast Growth Factor Mutein

Abstract

During large scale preparation of recombinant human basic fibroblast growth factor (hbFGF) mutein CS23(CS23) produced in Escherichia coli (E. coli), three species, named Mf-1, Mf-2 and Mf-3 in the order of their elution, were isolated from the early fraction of Sulfated Cellulofine chromatography by high performence liquid chromatography (HPLC) on heparin-5pw. Structural analysis through HPLC peptide mapping, mass spectrometry, sequencing and amino acid composition revealed that epsilon-N-acetylation occurred at positions Lys135, Lys26 and Lys119 of CS23. Together with this observation, the results on large scale preparation of CS23 were also presented.

Bacterial protein acetylation: new discoveries unanswered questions

“…Lysine acetylation has been detected following production in E. coli of recombinant therapeutic proteins, including the chemokine RANTES (d'Alayer et al, 2007), human basic fibroblast growth factor mutein (Suenaga et al, 1996), human carbonic anhydrase (Mahon et al, 2015), insulin lispro (Szewczak et al, 2015), interleukin-10 (Pflumm et al, 1997), interleukin-2 (Moya et al, 2002), interferon alpha (Takao et al, 1987), neurotropin-3 (Ross et al, 1996), and somatotropin (Violand et al, 1994). Nearly 30% of currently approved recombinant therapeutic proteins are produced in E. coli (Huang et al, 2012).…”

Choice of Cellular Protein Expression System

“…This modification has been demonstrated in E. coli in expressing recombinant proteins, although it has been shown that these adventitious post‐translational modifications are mostly undesirable. A recombinant human basic fibroblast growth factor mutein, expressed in E. coli , exhibited ɛ‐N‐acetylation of three lysines, although there is no apparent functionality associated with this modification (Suenaga et al, ). In epithelial cells, keratin undergoes a number of post‐transcriptional modifications, including acetylation of the N‐terminal serine (Omary et al, ).…”

Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli

“…AcP is a small-molecule metabolite in the phosphotransacetylase (Pta)—acetate kinase (AckA) pathway, one of the basic metabolic networks in E. coli ( 19 , 21 ). In light of the recent explosion of basic research on lysine acetylation ( 22 – 24 ), it seems important to recall previous studies on acetylation of recombinant therapeutic proteins produced in E. coli : neurotropin-3 ( 25 ), somatotropin ( 16 ), interleukin-10 ( 26 ), interleukin-2 ( 27 ), chemokine RANTES (regulated on activation, normal T cell expressed and secreted) ( 17 ), interferon alfa ( 28 ) and gamma ( 29 ), stathmin-like subdomains ( 30 ), human basic fibroblast growth factor mutein ( 31 ) and prochymosin ( 32 ). These results support current attempts to understand the mechanism of acetylation and its implications on cell life.…”