Intrabody construction and expression. I. The critical role of VL domain stability.


Abstract

We have constructed a panel of hyperstable immunoglobulin VL domains by a rational approach of consensus sequence engineering and combining stabilizing point mutations. These prototype domains unfold fully reversibly, even when the conserved structural disulfide bridge is reduced. This has allowed us to probe the factors that limit the expression yield of soluble immunoglobulin domains in the reducing environment of the cytoplasm (intrabodies). The most important factor is thermodynamic stability, and there is an excellent quantitative correlation between stability and yield. Surprisingly, an unprocessed N-terminal methionine residue can severely compromise VL stability, but this problem can be overcome by changing the amino acid following the initiator methionine residue. Transcription from the strong T7 promoter does not increase the amount of soluble material over that obtained from the tetA promoter, but large amounts of inclusions bodies can be obtained. Elevated temperature shifts protein from a productive folding pathway to aggregation. The structural disulfide bridge does not form in the cytoplasm, but the two consensus cysteine residues can be quantitatively oxidized in vitro. In summary, stability engineering provides a plannable route to the high-yield cytoplasmic expression of functional intrabody domains. Study holds ProTherm entries: 6068, 6069, 6070, 6071, 6072, 6073, 6074, 6075, 6076 Extra Details: additive : EDTA(20 mM),oxidized intrabodies; recombinant expression; VL domain;,protein stability; protein engineering

Submission Details

ID: vkjsHhys3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Ohage E;Steipe B,J. Mol. Biol. (1999) Intrabody construction and expression. I. The critical role of VL domain stability. PMID:10518947
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2ATY 2006-01-31 Complement receptor chimaeric conjugate CR2-Ig
1P7K 2004-05-11 1.75 Crystal structure of an anti-ssDNA antigen-binding fragment (Fab) bound to 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)
1SEQ 2005-03-01 1.78 Fab MNAC13
1KEL 1996-12-07 1.9 CATALYTIC ANTIBODY 28B4 FAB FRAGMENT COMPLEXED WITH HAPTEN (1-[N-4'-NITROBENZYL-N-4'-CARBOXYBUTYLAMINO] METHYLPHOSPHONIC ACID)
1ORS 2003-05-06 1.9 X-ray structure of the KvAP potassium channel voltage sensor in complex with an Fab
1R3J 2003-11-25 1.9 potassium channel KcsA-Fab complex in high concentration of Tl+
1GPO 1998-04-01 1.95 CRYSTAL STRUCTURE OF THE RATIONALLY DESIGNED ANTIBODY M41 AS A FAB FRAGMENT
1A3L 1999-02-16 1.95 CATALYSIS OF A DISFAVORED REACTION: AN ANTIBODY EXO DIELS-ALDERASE-TSA-INHIBITOR COMPLEX AT 1.95 A RESOLUTION
1OPG 1995-07-31 2.0 OPG2 FAB FRAGMENT
1P2C 2004-02-17 2.0 crystal structure analysis of an anti-lysozyme antibody
1BM3 1999-04-20 2.0 IMMUNOGLOBULIN OPG2 FAB-PEPTIDE COMPLEX
25C8 1999-03-23 2.0 CATALYTIC ANTIBODY 5C8, FAB-HAPTEN COMPLEX
1FNS 2000-10-18 2.0 CRYSTAL STRUCTURE OF THE VON WILLEBRAND FACTOR (VWF) A1 DOMAIN I546V MUTANT IN COMPLEX WITH THE FUNCTION BLOCKING FAB NMC4
1CLO 1997-05-15 2.1 ANTI-CARCINOEMBRYONIC ANTIGEN MONOCLONAL ANTIBODY A5B7
1CT8 1999-11-10 2.2 CATALYTIC ANTIBODY 7C8 COMPLEX
1KCU 2002-05-11 2.2 CRYSTAL STRUCTURE OF ANTIBODY PC287
1OAK 1998-10-21 2.2 CRYSTAL STRUCTURE OF THE VON WILLEBRAND FACTOR (VWF) A1 DOMAIN IN COMPLEX WITH THE FUNCTION BLOCKING NMC-4 FAB
1KEM 1996-12-07 2.2 CATALYTIC ANTIBODY 28B4 FAB FRAGMENT
1EMT 2000-11-01 2.25 FAB ANTIBODY FRAGMENT OF AN C60 ANTIFULLERENE ANTIBODY
1A0Q 1999-03-02 2.3 29G11 COMPLEXED WITH PHENYL [1-(1-N-SUCCINYLAMINO)PENTYL] PHOSPHONATE
1K4D 2001-11-14 2.3 Potassium Channel KcsA-Fab complex in low concentration of K+
1R3I 2003-11-25 2.4 potassium channel KcsA-Fab complex in Rb+
1R3L 2003-11-25 2.41 potassium channel KcsA-Fab complex in Cs+
1CIC 1999-04-19 2.5 IDIOTOPE-ANTI-IDIOTOPE FAB-FAB COMPLEX; D1.3-E225
1CK0 2003-08-05 2.5 ANTI-ANTI-IDIOTYPIC ANTIBODY AGAINST HUMAN ANGIOTENSIN II, UNLIGANDED FORM
1KC5 2002-07-24 2.5 CRYSTAL STRUCTURE OF ANTIBODY PC287 IN COMPLEX WITH PS1 PEPTIDE
1RIH 2004-01-13 2.5 Crystal Structure of Fab 14F7, a unique anti-tumor antibody specific for N-glycolyl GM3
1FDL 1991-10-15 2.5 CRYSTALLOGRAPHIC REFINEMENT OF THE THREE-DIMENSIONAL STRUCTURE OF THE FAB D1.3-LYSOZYME COMPLEX AT 2.5-ANGSTROMS RESOLUTION
1MLC 1995-06-03 2.5 MONOCLONAL ANTIBODY FAB D44.1 RAISED AGAINST CHICKEN EGG-WHITE LYSOZYME COMPLEXED WITH LYSOZYME
15C8 1999-03-23 2.5 CATALYTIC ANTIBODY 5C8, FREE FAB
1MF2 1997-12-31 2.6 ANTI HIV1 PROTEASE FAB COMPLEX
1C12 1999-08-14 2.6 INSIGHT IN ODORANT PERCEPTION: THE CRYSTAL STRUCTURE AND BINDING CHARACTERISTICS OF ANTIBODY FRAGMENTS DIRECTED AGAINST THE MUSK ODORANT TRASEOLIDE
1DBJ 1994-01-31 2.7 MOLECULAR BASIS OF CROSS-REACTIVITY AND THE LIMITS OF ANTIBODY-ANTIGEN COMPLEMENTARITY
1DBM 1994-01-31 2.7 MOLECULAR BASIS OF CROSS-REACTIVITY AND THE LIMITS OF ANTIBODY-ANTIGEN COMPLEMENTARITY
1S5I 2004-05-25 2.7 Fab (LNKB-2) of monoclonal antibody to Human Interleukin-2, crystal structure
1DBB 1993-10-31 2.7 THREE-DIMENSIONAL STRUCTURE OF AN ANTI-STEROID FAB' AND PROGESTERONE-FAB' COMPLEX
1R0A 2004-08-03 2.8 Crystal structure of HIV-1 reverse transcriptase covalently tethered to DNA template-primer solved to 2.8 angstroms
1AI1 1997-05-15 2.8 HIV-1 V3 LOOP MIMIC
1JRH 1998-03-25 2.8 COMPLEX (ANTIBODY/ANTIGEN)
1QFU 1999-04-16 2.8 INFLUENZA VIRUS HEMAGGLUTININ COMPLEXED WITH A NEUTRALIZING ANTIBODY
1R3K 2003-11-25 2.8 potassium channel KcsA-Fab complex in low concentration of Tl+
1DBA 1993-10-31 2.8 THREE-DIMENSIONAL STRUCTURE OF AN ANTI-STEROID FAB' AND PROGESTERONE-FAB' COMPLEX
2DBL 1994-03-07 2.9 MOLECULAR BASIS OF CROSS-REACTIVITY AND THE LIMITS OF ANTIBODY-ANTIGEN COMPLEMENTARITY
1KCR 2002-05-11 2.9 CRYSTAL STRUCTURE OF ANTIBODY PC283 IN COMPLEX WITH PS1 PEPTIDE
1CL7 2000-01-12 3.0 ANTI HIV1 PROTEASE FAB
1DBK 1994-01-31 3.0 MOLECULAR BASIS OF CROSS-REACTIVITY AND THE LIMITS OF ANTIBODY-ANTIGEN COMPLEMENTARITY
1N6Q 2003-01-14 3.0 HIV-1 Reverse Transcriptase Crosslinked to pre-translocation AZTMP-terminated DNA (complex N)
1FBI 1995-02-27 3.0 CRYSTAL STRUCTURE OF A CROSS-REACTION COMPLEX BETWEEN FAB F9.13.7 AND GUINEA-FOWL LYSOZYME
1ACY 1994-07-31 3.0 CRYSTAL STRUCTURE OF THE PRINCIPAL NEUTRALIZING SITE OF HIV-1
1N5Y 2003-01-28 3.1 HIV-1 Reverse Transcriptase Crosslinked to Post-Translocation AZTMP-Terminated DNA (Complex P)
1YED 1997-10-15 3.1 STRUCTURE OF A CATALYTIC ANTIBODY IGG2A FAB FRAGMENT (D2.4)
1IGY 1998-04-15 3.2 STRUCTURE OF IMMUNOGLOBULIN
1KEN 2002-04-24 3.5 INFLUENZA VIRUS HEMAGGLUTININ COMPLEXED WITH AN ANTIBODY THAT PREVENTS THE HEMAGGLUTININ LOW PH FUSOGENIC TRANSITION
1NTL 2004-02-03 30.0 Model of mouse Crry-Ig determined by solution scattering, curve fitting and homology modelling

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ig gamma-1 chain C region secreted form P01869 IGH1M_MOUSE
100.0 Ig gamma-1 chain C region secreted form P01868 IGHG1_MOUSE