Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain

Kaija Alfthan (Corresponding Author), Kristiina Takkinen, Dorothea Sizmann, Ilari Seppälä, Tiina Immonen, Liisa Vanne, Sirkka Keränen, Matti Kaartinen, Jonathan Knowles, Tuula Teeri

Research output: Contribution to journalArticleScientificpeer-review

30 Citations (Scopus)

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 μg/ml and 0.5 μg/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
DOIs
Publication statusPublished - 1993
MoE publication typeA1 Journal article-refereed

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Immunoglobulin Fab Fragments
Immunoglobulin G
Escherichia coli
Culture Media
Periplasm
Antibodies
Haptens
Protein Sorting Signals
Affinity Chromatography
Disulfides
Hot Temperature
Monoclonal Antibodies

Cite this

Alfthan, Kaija ; Takkinen, Kristiina ; Sizmann, Dorothea ; Seppälä, Ilari ; Immonen, Tiina ; Vanne, Liisa ; Keränen, Sirkka ; Kaartinen, Matti ; Knowles, Jonathan ; Teeri, Tuula. / Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain. In: Gene. 1993 ; Vol. 128, No. 2. pp. 203 - 209.
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title = "Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain",
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 μg/ml and 0.5 μg/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.",
author = "Kaija Alfthan and Kristiina Takkinen and Dorothea Sizmann and Ilari Sepp{\"a}l{\"a} and Tiina Immonen and Liisa Vanne and Sirkka Ker{\"a}nen and Matti Kaartinen and Jonathan Knowles and Tuula Teeri",
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doi = "10.1016/0378-1119(93)90564-J",
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pages = "203 -- 209",
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Alfthan, K, Takkinen, K, Sizmann, D, Seppälä, I, Immonen, T, Vanne, L, Keränen, S, Kaartinen, M, Knowles, J & Teeri, T 1993, 'Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain', Gene, vol. 128, no. 2, pp. 203 - 209. https://doi.org/10.1016/0378-1119(93)90564-J

Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain. / Alfthan, Kaija (Corresponding Author); Takkinen, Kristiina; Sizmann, Dorothea; Seppälä, Ilari; Immonen, Tiina; Vanne, Liisa; Keränen, Sirkka; Kaartinen, Matti; Knowles, Jonathan; Teeri, Tuula.

In: Gene, Vol. 128, No. 2, 1993, p. 203 - 209.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain

AU - Alfthan, Kaija

AU - Takkinen, Kristiina

AU - Sizmann, Dorothea

AU - Seppälä, Ilari

AU - Immonen, Tiina

AU - Vanne, Liisa

AU - Keränen, Sirkka

AU - Kaartinen, Matti

AU - Knowles, Jonathan

AU - Teeri, Tuula

N1 - Project code: BIO8015

PY - 1993

Y1 - 1993

N2 - 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 μg/ml and 0.5 μg/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.

AB - 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 μg/ml and 0.5 μg/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.

U2 - 10.1016/0378-1119(93)90564-J

DO - 10.1016/0378-1119(93)90564-J

M3 - Article

VL - 128

SP - 203

EP - 209

JO - Gene

JF - Gene

SN - 0378-1119

IS - 2

ER -