EFFICIENT SECRETION OF MURINE FAB FRAGMENTS BY ESCHERICHIA-COLI IS DETERMINED BY THE 1ST CONSTANT DOMAIN OF THE HEAVY-CHAIN

K ALFTHAN*, K TAKKINEN, D SIZMANN, Ilkka Seppälä, T IMMONEN, L VANNE, S KERANEN, M KAARTINEN, JKC KNOWLES, Tuula T. Teeri

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 mug/ml and 0.5 mug/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.

Original languageEnglish
Pages (from-to)203-209
Number of pages7
JournalGene
Volume128
Issue number2
Publication statusPublished - 30 Jun 1993
MoE publication typeA1 Journal article-refereed

Keywords

  • PERIPLASMIC SPACE
  • 2-PHENYLOXAZOLONE
  • RECOMBINANT ANTIBODY
  • ASSEMBLY AND EXPRESSION
  • STABILITY
  • B O-ANTIGEN
  • BACTERIAL EXPRESSION
  • ANTIBODY FRAGMENTS
  • MESSENGER-RNA
  • PROTEINS
  • FV
  • PURIFICATION
  • GROWTH
  • GENES
  • CELLS

Cite this