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Isolation of Monomeric Human VHS by a Phage Selection

2005; Elsevier BV; Volume: 280; Issue: 50 Linguagem: Inglês

10.1074/jbc.m509900200

1083-351X

Rebecca To, Tomoko Hirama, Mehdi Arbabi‐Ghahroudi, Roger MacKenzie, Ping Wang, Ping Xu, Feng Ni, Jamshid Tanha,

Protein purification and stability

Human VH domains are promising molecules in applications involving antibodies, in particular, immunotherapy because of their human origin. However, they are, in general, prone to aggregation. Therefore, various strategies have been employed to acquire monomeric human VHs. We had previously discovered that filamentous phages displaying engineered monomeric VH domains gave rise to significantly larger plaques on bacterial lawns than phages displaying wild type VHs with aggregation tendencies. Using plaque size as the selection criterion and a phage-displayed naïve human VH library we identified 15 VHs that were monomeric. Additionally, the VHs demonstrated good expression yields, good refolding properties following thermal denaturation, resistance to aggregation during long incubation at 37 °C, and to trypsin at 37 °C. These 15 VHs should serve as good scaffolds for developing immunotherapeutics, and the selection method employed here should have general utility for isolating proteins with desirable biophysical properties. Human VH domains are promising molecules in applications involving antibodies, in particular, immunotherapy because of their human origin. However, they are, in general, prone to aggregation. Therefore, various strategies have been employed to acquire monomeric human VHs. We had previously discovered that filamentous phages displaying engineered monomeric VH domains gave rise to significantly larger plaques on bacterial lawns than phages displaying wild type VHs with aggregation tendencies. Using plaque size as the selection criterion and a phage-displayed naïve human VH library we identified 15 VHs that were monomeric. Additionally, the VHs demonstrated good expression yields, good refolding properties following thermal denaturation, resistance to aggregation during long incubation at 37 °C, and to trypsin at 37 °C. These 15 VHs should serve as good scaffolds for developing immunotherapeutics, and the selection method employed here should have general utility for isolating proteins with desirable biophysical properties. The immune repertoire of Camelidae is unique in that it possesses unusual types of antibodies referred to as heavy chain antibodies (1Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2100) Google Scholar). These antibodies lack light chains and thus their combining sites consist of only one domain, termed VHH. 2The abbreviations used are: VHclassical four-chain antibody heavy chain variable domainVHHvariable domain of a heavy chain antibodyCDRcomplementarity determining regionCHheavy chain constant domain of an antibodyFabantigen-binding fragmentFRframework regionNMRnuclear magnetic resonancePFGpulse field gradientRErefolding efficiencyscFvsingle chain Fv fragment of an antibodysdAbsingle domain antibodySPRsurface plasmon resonanceVLantibody light chain variable domain.2The abbreviations used are: VHclassical four-chain antibody heavy chain variable domainVHHvariable domain of a heavy chain antibodyCDRcomplementarity determining regionCHheavy chain constant domain of an antibodyFabantigen-binding fragmentFRframework regionNMRnuclear magnetic resonancePFGpulse field gradientRErefolding efficiencyscFvsingle chain Fv fragment of an antibodysdAbsingle domain antibodySPRsurface plasmon resonanceVLantibody light chain variable domain. Recombinant VHH single-domain antibodies (sdAbs) are comparable with their single-chain Fv (scFv) counterparts derived from conventional four-chain antibodies in terms of affinity, but outperform scFvs in terms of stability, resistance to aggregation, refolding properties, expression yield, and relative ease of DNA manipulation, library construction, and three-dimensional structural determination (2van der Linden R.H. Frenken L.G. de Geus B. Harmsen M.M. Ruuls R.C. Stok W. de Ron L. Wilson S. Davis P. Verrips C.T. Biochim. Biophys. Acta. 1999; 1431: 37-46Crossref PubMed Scopus (346) Google Scholar, 3Frenken L.G. van der Linden R.H. Hermans P.W. Bos J.W. Ruuls R.C. de Geus B. Verrips C.T. J. Biotechnol. 2000; 78: 11-21Crossref PubMed Scopus (225) Google Scholar, 4Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (129) Google Scholar, 5Tanha J. Dubuc G. Hirama T. Narang S.A. MacKenzie C.R. J. Immunol. Methods. 2002; 263: 97-109Crossref PubMed Scopus (111) Google Scholar, 6Zhang J. Tanha J. Hirama T. Khieu N.H. To R. Tong-Sevinc H. Stone E. Brisson J.R. MacKenzie C.R. J. Mol. Biol. 2004; 335: 49-56Crossref PubMed Scopus (148) Google Scholar). Many of the aforementioned properties of VHH sdAbs are desired in applications involving antibodies. However, the non-human nature of VHHs limits their use in human immunotherapy because of the immunogenicity issue. In this respect, human VH sdAbs are ideal candidates for therapeutic applications because they are expected to be least immunogenic. However, human VHs are by and large prone to aggregation, a characteristic common to VHs derived from conventional antibodies (7Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (143) Google Scholar, 8Tanha J. Xu P. Chen Z.G. Ni F. Kaplan H. Narang S.A. MacKenzie C.R. J. Biol. Chem. 2001; 276: 24774-24780Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 9Ward E.S. Gussow D. Griffiths A.D. Jones P.T. Winter G. Nature. 1989; 341: 544-546Crossref PubMed Scopus (885) Google Scholar). Thus, attempts were made previously to obtain monomers, i.e. human VHs suitable for antibody applications. Such VHs also displayed other useful properties typical of VHHs, such as high expression yield, high refoldability, and resistance to aggregation. Synthetic libraries built on these VHs as library scaffolds should serve as a promising source of therapeutic proteins.Camelization (7Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (143) Google Scholar, 8Tanha J. Xu P. Chen Z.G. Ni F. Kaplan H. Narang S.A. MacKenzie C.R. J. Biol. Chem. 2001; 276: 24774-24780Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) as well as llamination, 3J. Tanha, T.-D. Nguyen, A. Ng, F. Ni, and C. R. MacKenzie, manuscript in preparation.3J. Tanha, T.-D. Nguyen, A. Ng, F. Ni, and C. R. MacKenzie, manuscript in preparation. which involves incorporating key solubilizing residues from camelid sdAbs into human VHs, have been employed to generate monomeric human VHs. Synthetic sdAb libraries constructed based on these VHs and generated by complementarity determining region (CDR) randomization were shown to yield binders to various antigens (8Tanha J. Xu P. Chen Z.G. Ni F. Kaplan H. Narang S.A. MacKenzie C.R. J. Biol. Chem. 2001; 276: 24774-24780Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Davies J. Riechmann L. Biotechnology. 1995; 13: 475-479Crossref PubMed Scopus (134) Google Scholar). In another approach, fully monomeric human VHs were isolated from human synthetic VH libraries without resorting to engineering of the sort mentioned above. In one experiment a monomeric human VH was discovered when a human VH library was panned against hen egg white lysozyme (11Jespers L. Schon O. James L.C. Veprintsev D. Winter G. J. Mol. Biol. 2004; 337: 893-903Crossref PubMed Scopus (101) Google Scholar). More recently, a selection method based on reversible unfolding and affinity criteria yielded many monomeric VHs from synthetic human VH phage display libraries (12Jespers L. Schon O. Famm K. Winter G. Nat. Biotechnol. 2004; 22: 1161-1165Crossref PubMed Scopus (228) Google Scholar). This finding underlined the fact that an appropriate selection method is key to efficient capturing of rare human VHs with desirable biophysical properties.Here, we provide yet another approach for obtaining monomeric human VHs. We report the isolation of 15 different VHs originating from germlines DP-38, DP-47, V3-49, V3-53, YAC-5, and 8-1B from a phage-displayed naïve human VH repertoire by a selection method that is based on phage plaque size. The VHs, by and large, are also refoldable, retain their native fold following exposure to trypsin at 37 °C or long incubation at 37 °C, and are expressed in good yields in the Escherichia coli. When used as scaffolds, the diversity of the selected VHs should allow for construction of more comprehensive libraries and provide flexibility in terms of choosing an optimal VH scaffold for humanizing therapeutic camelid VHH binders. The current selection method permits high throughput identification of proteins with good biophysical properties by the naked eye, is very simple, eliminates affinity or stability selection steps, and is of general utility.MATERIALS AND METHODSPhage Display Library Construction and Panning—cDNA was synthesized from human spleen mRNA (Ambion Inc., Austin, TX) using random hexanucleotide primers and First Strand cDNA® kit (GE Healthcare, Baie d'Urfé, QC, Canada). Using the cDNA as template, VH genes with flanking CH sequences were amplified by polymerase chain reaction in nine separate reactions using VH framework region 1 (FR1)-specific primers and an immunoglobulin M-specific primer (13de Haard H.J. van Neer N. Reurs A. Hufton S.E. Roovers R.C. Henderikx P. de Bruine A.P. Arends J.W. Hoogenboom H.R. J. Biol. Chem. 1999; 274: 18218-18230Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). The products were gel-purified and used as the template in the second round of PCR to construct VH genes using the FR1- and FR4-specific primers (13de Haard H.J. van Neer N. Reurs A. Hufton S.E. Roovers R.C. Henderikx P. de Bruine A.P. Arends J.W. Hoogenboom H.R. J. Biol. Chem. 1999; 274: 18218-18230Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar) that also introduced flanking ApalI and NotI restriction sites for cloning purposes. The resultant VH repertoire DNA was cloned into fd-tetGIIID phage vector and a VH phage display library was constructed (8Tanha J. Xu P. Chen Z.G. Ni F. Kaplan H. Narang S.A. MacKenzie C.R. J. Biol. Chem. 2001; 276: 24774-24780Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Panning against protein A (GE Healthcare) was performed as described (8Tanha J. Xu P. Chen Z.G. Ni F. Kaplan H. Narang S.A. MacKenzie C.R. J. Biol. Chem. 2001; 276: 24774-24780Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Germline sequence assignment of the selected VHswas performed using DNAPLOT software version 2.0.1 and V BASE version 1.0. 4vbase.dnaplot.de/cgi-bin/vbase/vsearch.pl. Llama VHHs H11C7, H11F9, and H11B2 were isolated from a llama VHH phage display library by panning against H11 scFv as described (5Tanha J. Dubuc G. Hirama T. Narang S.A. MacKenzie C.R. J. Immunol. Methods. 2002; 263: 97-109Crossref PubMed Scopus (111) Google Scholar).Protein Expression and Purification—Single-domain antibodies were cloned into pSJF2 expression vector by standard cloning techniques (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. 1989; (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)Google Scholar). Periplasmic expression of sdAbs and subsequent purification by immobilized metal affinity chromatography were performed as described (15Tanha J. Muruganandam A. Stanimirovic D. Methods Mol. Med. 2003; 89: 435-450PubMed Google Scholar). Protein concentrations were determined by A280 measurements using molar absorption coefficients calculated for each protein (16Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3395) Google Scholar). Gel filtration chromatography of the purified sdAbs was performed on a Superdex 75 column (GE Healthcare) as described (17Deng S.J. MacKenzie C.R. Hirama T. Brousseau R. Lowary T.L. Young N.M. Bundle D.R. Narang S.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4992-4996Crossref PubMed Scopus (53) Google Scholar).Binding and Refolding Efficiency Experiments—Equilibrium dissociation constants (KDs) and refolding efficiencies (REs) of VHs/VHHs were derived from surface plasmon resonance (SPR) data collected with the BIACORE 3000 biosensor system (Biacore Inc., Piscataway, NJ). To measure the binding of VHs to protein A, 2000 resonance units of protein A or a reference antigen-binding fragment (Fab) were immobilized on research grade CM5 sensor chips (Biacore Inc.). Immobilizations were carried out at concentrations of 25 μg/ml (protein A) or 50 μg/ml (Fab) in 10 mm sodium acetate buffer, pH 4.5, using the amine coupling kit provided by the manufacturer. To measure the binding of the anti-idiotypic llama VHHs to H11 scFv (18Reilly R.M. Maiti P.K. Kiarash R. Prashar A.K. Fast D.G. Entwistle J. Narang Dan Foote S.A. Kaplan S.H.A. Nucleic Med. Commun. 2001; 22: 587-595Crossref PubMed Scopus (10) Google Scholar), 4100 resonance units of 50 μg/ml H11 scFv or 3000 resonance units of 10 μg/ml Se155-4 immunoglobulin G (19Bundle D.R. Eichler E. Gidney M.A. Meldal M. Ragauskas A. Sigurskjold B.W. Sinnott B. Watson D.C. Yaguchi M. Young N.M. Biochemistry. 1994; 33: 5172-5182Crossref PubMed Scopus (92) Google Scholar) were immobilized as described above. In all instances, analyses were carried out at 25 °C in 10 mm HEPES, pH 7.4, containing 150 mm NaCl, 3 mm EDTA, and 0.005% P20 surfactant at a flow rate of 40 μl/min, and surfaces were regenerated by washing with the running buffer. To determine the binding activities of the refolded proteins, VHs or VHHs were denatured by incubation at 85 °C for 20 min at 10 μg/ml concentrations. The protein samples were then cooled down to room temperature for 30 min to refold and were subsequently centrifuged in a microcentrifuge at 14,000 × g for 5 min at room temperature to remove any protein precipitates. The supernatants were recovered and analyzed for binding activity by SPR as described above. For both folded and refolded protein, data were fit to a 1:1 interaction model simultaneously using BIAevaluation 4.1 software (Biacore Inc.) and KD values were subsequently determined. REs were determined from RE = (KDn/KDref) × 100, where KDn is the KD of the native protein and KDref is the KD of the refolded protein.Tryptic Digest Experiments—Three μl of a freshly prepared 0.1 μg/μl sequencing grade trypsin (Hoffmann-La Roche Ltd., Mississauga, ON, Canada) in 1 mm HCl was added to 60 μg of VH in 100 mm Tris-HCl buffer, pH 7.8. Digestion reactions were carried out in a total volume of 60 μl for 1 h at 37°C and stopped by adding 5 μl of 0.1 μg/μl trypsin inhibitor (Sigma). Following completion of digestion, 5 μl was removed and analyzed by SDS-PAGE; the remaining was desalted using ZipTipC4 (Millipore, Ontario, Canada), eluted with 1% acetic acid in 50:50 methanol:water and subjected to VH mass determination by matrix-assisted laser desorption ionization mass spectrometry.Protein Stability Experiments at 37 °C—Single-domain antibodies at 0.32–3.2 mg/ml concentrations were incubated at 37 °C in phosphate-buffered saline buffer for 17 days. Following incubation, the protein samples were spun down in a microcentrifuge at maximum speed for 5 min even in the absence of any visible aggregate formation. The samples were then applied onto a Superdex 75 size exclusion column and the monomeric peaks were collected for SPR analysis of binding to protein A. SPR analyses were performed as described above except that 500 resonance units of protein A or reference Fab was immobilized and immobilizations were carried out at a concentration of 50 μg/ml.NMR Experiments—VH samples for NMR analysis were dissolved in 10 mm sodium phosphate, 150 mm NaCl, 0.5 mm EDTA, and 0.02% NaN3 at pH 7.0. The protein concentrations were 40 μm to 1.0 mm. All NMR experiments were carried out at 298 K on a Bruker Avance-800 or a Bruker Avance-500 NMR spectrometer. One-dimensional 1H NMR spectra were recorded with 16,384 data points and the spectral widths were 8,992.81 Hz at 500 MHz and 17,605.63 Hz at 800 MHz, respectively. Two-dimensional 1H-1H NOESY spectra of 2,048 × 400 data points were acquired on a Bruker Avance-800 NMR spectrometer with a spectral width of 11,990.04 Hz and a mixing time of 120 ms. In all NMR experiments, water suppression was achieved using the WATERGATE method implemented through the 3-9-19 pulse train (20Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3499) Google Scholar, 21Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. 1993; A102: 241-245Crossref Scopus (1108) Google Scholar). NMR data were processed and analyzed using the Bruker XWINNMR software package. All PFG-NMR diffusion measurements were carried out with the water-suppressed LED sequence (22Altieri A.S. Hilton D.P. Byrd R.A. J. Am. Chem. Soc. 1995; 117: 7566-7567Crossref Scopus (437) Google Scholar), on a Bruker Avance-500 NMR spectrometer equipped with a triple-resonance probe with three-axis gradients. One-dimensional proton spectra were processed and analyzed using Bruker Xwinnmr software package. NMR signal intensities were obtained by integrating NMR spectra in the methyl and methylene proton region (2.3 to -0.3 ppm) where all NMR signals were attenuated uniformly at all given PFG strengths.RESULTSDuring the course of the construction of fully human and llaminated human VH libraries,3 we learned that the phages displaying monomeric llaminated VHs formed larger plaques on bacterial lawns than phages displaying fully human VHs with aggregation tendencies. We thus decided to use plaque size as a means of identifying rare, naturally occurring monomer VHs from the human VH repertoire (Fig. 1). To this end, a phage library displaying human VHs with a size of 6 × 108 was constructed and propagated as plaques on agar plates. On the titer plates, the library consisted essentially of small plaques interspersed with some large ones. PCR on 20 clones revealed that the small plaques corresponded to the VH-displaying phages, whereas the large ones represented the wild type phages, i.e. phages lacking VH sequence inserts. None of the VH-displaying phages were found with large plaque morphology. This was not unexpected because of the paucity of the monomeric VHs in the human repertoire and the large size of the library. To facilitate the identification of monomeric VHs, it was decided to reduce the library size to a manageable one and remove interfering wild type phage with large plaque-size morphology by panning the library against protein A, which binds to a subset of human VHs from the VH3 family.Following a few rounds of panning, the library became enriched for phage producing large plaques, and PCR and sequencing of more than 110 such plaques showed that all had complete VH open reading frames. The size of the large plaques that were picked for analysis is represented in Fig. 1. Sequencing revealed 15 different VHs that belonged to the VH3 family and utilized DP-38, DP-47, V3-49, V3-53, YAC-5, or 8-1B germline V segments (TABLE ONE; Fig. 2). The DP-38 and DP-47 germline sequences have been previously implicated in protein A binding (12Jespers L. Schon O. Famm K. Winter G. Nat. Biotechnol. 2004; 22: 1161-1165Crossref PubMed Scopus (228) Google Scholar, 23Akerstrom B. Nilson B.H. Hoogenboom H.R. Bjorck L. J. Immunol. Methods. 1994; 177: 151-163Crossref PubMed Scopus (41) Google Scholar). In addition, all VHs had a Thr residue at position 57 (Fig. 2), consistent with their protein A binding activity (24Bond C.J. Marsters J.C. Sidhu S.S. J. Mol. Biol. 2003; 332: 643-655Crossref PubMed Scopus (75) Google Scholar, 25Randen I. Potter K.N. Li Y. Thompson K.M. Pascual V. Forre O. Natvig J.B. Capra J.D. Eur. J. Immunol. 1993; 23: 2682-2686Crossref PubMed Scopus (50) Google Scholar). The most frequently utilized germline V segment was DP-47, which occurred in over 50% of the VHs, but the most frequent clone (i.e. HVHP428, relative frequency 46%) utilized the V3-49 germline V segment. HVHP429 with a DP-47 germline sequence was the second most abundant VH with a relative frequency of 21% (Fig. 2). The VH CDR3 lengths ranged from 4 amino acids for HVHB82 to 16 amino acids for HVHP430, with HVHP430 having a pair of Cys residues in CDR3. Amino acid mutations with respect to the parental germline V segment (residues 1–94) and FR4 (residues 103–113) sequences were observed in all VHs and ranged from two mutations for HVHP44 (L5V and Q105R) and HVHB82 (E1Q and L5Q) to 16 mutations for HVHP426 (TABLE ONE). Mutations were concentrated in the V segments; only two mutations were detected in all the 15 FR4s, at positions 105 and 108. HVHP44 and HVHB82 differed from other VHs in that they both had a positively charged amino acid at position 105 instead of a Gln (TABLE ONE, Fig. 2). However, whereas the positively charged amino acid in HVHP44 was acquired by mutation, the one in HVHB82 was germline-encoded. Except for HVHP423 and HVHP44B, the remaining VHs had the germline residues at the key solubility positions (4Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-296Crossref PubMed Scopus (129) Google Scholar): 37V/44G/45L/47W or 37F/44G/45L/47W (HVHP428), HVHP423 and HVHP44B had a V37F mutation. Mutations at other positions, which have been shown or hypothesized to be important in VH solubility, included seven E6Q, three S35T/H, one R83G, one K83R, one A84P, one T84A, and one M108L mutation (5Tanha J. Dubuc G. Hirama T. Narang S.A. MacKenzie C.R. J. Immunol. Methods. 2002; 263: 97-109Crossref PubMed Scopus (111) Google Scholar, 11Jespers L. Schon O. James L.C. Veprintsev D. Winter G. J. Mol. Biol. 2004; 337: 893-903Crossref PubMed Scopus (101) Google Scholar, 26Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. Nat. Struct. Biol. 1996; 3: 752-757Crossref PubMed Scopus (130) Google Scholar). Frequent mutations were also observed at positions 1 and 5 that included 11 E1Q, eight L5V/Q, and one V5Q mutations.TABLE ONEVH sequence deviations from parental germline sequencesVHV/J germlineAmino acid deviation from V and FR4 germline sequencesHVHP44DP47/JH4bL5V, Q105RHVHB82DP47/JH6cE1Q, L5QHVHP421DP47/JH4bE1Q, V2L, L5Q, L11V, G16RHVHP419DP47/JH4bE1Q, V2L, L5Q, T77S, R83G, K94RHVHP430DP47/JH3bE1Q, L5V, V12I, Q13K, S31N, G52AS, L78V, A93V, K94RHVHP429DP47/JH4L5V, G10T, S30I, S31N, G42D, E46D, A50T, G52aN, S53N, S56A K75N, A84P, E85DHVHM41DP47/JH3aE1Q, L5V, E6Q, G16R, T28A, S53G, G55D, S56H, M108LHVHM81DP47JH3aL5V, E6Q, G16R, S30D, S31D, S35H, A50G, G55A, E85G, V89L, K94RHVHP428V3–49/JH4bE1Q, V2L, V5Q, R16G, T23A, G30S, D31S, T60A, G73D, K83R, T84A, V89M, T93AHVHP420DP-38/JH4bE1Q, S35T, S52aTHVHP414DP-38/JH3bE1D, E6Q, A23T, T28P, K52T, A60VHVHP423V3–53/JH1E1Q, V2M, E6Q, L11V, I12V, N32S, Y33R, V37F, K43M, K64R, T68S, V89LHVHP44BV3–53/JH1E1Q, E6Q, N32S, Y33R, V37F, K43M, Y58S, K64R, T68S, V89LHVHP413YAC-5/JH3bE1Q, E6Q, Q13K, V29F, S31D, N32Y, V50FHVHP4268–1B/JH3bE1Q, E6Q, L11V, G16R, T28I, S30D, S31G, N32Y, Y33A, S35H, K43Q, I51T, Y52N, S53N, Y58S, L78V Open table in a new tab FIGURE 2Amino acid sequences of the human VHs selected based on affinity for protein A and plaque size. The dots in the sequence entries indicate amino acid identity with HVHP44. Dashes are included for sequence alignment. Residues at the key solubility positions and residue Thr57 that associates with VHs/VHHs protein A binding property are in bold. The Kabat numbering system is used (43). The total "frequency" value is 114.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All VHs except HVHP44B, which was essentially the same as HVHP423, were expressed in 1-liter culture volumes in E. coli strain TG1 in fusion with a c-Myc-His5 tag and purified to homogeneity from periplasmic extracts by immobilized metal affinity chromatography. The expression yields ranged from 1.8 to 62.1 mg of purified protein per liter of bacterial culture in shake flasks with the majority having yields of several milligrams (TABLE TWO). In the instances of HVHP423 and HVHP430, another trial under "apparently" the same expression conditions gave yields of 2.4 and 6.4 mg as opposed to 62.1 and 23.7 mg, respectively. This implies that for many of the VHs described here, optimal expression conditions should be achieved, without much effort, resulting in expression yields significantly higher than the values reported in TABLE TWO. As expected, all the VHs bound to protein A in SPR analyses, with KD values of 0.2–3 μm, a range and magnitude comparable with affinities reported previously for llama VHH variants with protein A binding activity (24Bond C.J. Marsters J.C. Sidhu S.S. J. Mol. Biol. 2003; 332: 643-655Crossref PubMed Scopus (75) Google Scholar). None of the VHs bound to the Fab reference surface.TABLE TWOBiophysical characteristics of the human VHsVH/VHHExpressionaExpression yield per liter of bacterial cultureKDTrypsin resistanceREOligomerization state (by GFC)bGFC, gel filtration chromatographyFolding and Oligomerization State (by NMR)cThe solution properties of the various VH molecules are characterized as soluble (s) and structurally folded (f) with broad (b), sharp (s), and partially sharp (ps) NMR spectramgμm%HVHP448.21.393Monomers/f/sdFolding was determined by one-dimensional 1H spectra recorded at 500 MHzHVHB825.90.271Monomers/f/sdFolding was determined by one-dimensional 1H spectra recorded at 500 MHzHVHP4215.51.014Monomers/f/seFolding was confirmed by one-dimensional 1H and two-dimensional 1H-1H NOESY spectra recorded at 800 MHz. No concentration dependent line broadenings were observed from ∼30 to 300 μmHVHP4193.41.684MonomerNDfND, not determinedHVHP4306.4, 23.72.388Monomers/f/psd,Folding was determined by one-dimensional 1H spectra recorded at 500 MHzgSharp resonances at a concentration of 40 μm and partial line-broadening were observed at ∼1.0 mmHVHP4293.41.386Monomers/f/seFolding was confirmed by one-dimensional 1H and two-dimensional 1H-1H NOESY spectra recorded at 800 MHz. No concentration dependent line broadenings were observed from ∼30 to 300 μmHVHM411.80.5X92MonomerNDHVHM814.31.387MonomerNDHVHP4283.11.895Monomers/f/seFolding was confirmed by one-dimensional 1H and two-dimensional 1H-1H NOESY spectra recorded at 800 MHz. No concentration dependent line broadenings were observed from ∼30 to 300 μmHVHP42059.01.292MonomerNDHVHP41411.81.673Monomers/f/shFolding was confirmed by one-dimensional 1H spectrum recorded at 800 MHzHVHP4232.4, 62.13.086Monomers/f/sdFolding was determined by one-dimensional 1H spectra recorded at 500 MHzHVHP4135.80.352Monomers/f/sdFolding was determined by one-dimensional 1H spectra recorded at 500 MHzHVHP4266.30.870Monomers/f/psdFolding was determined by one-dimensional 1H spectra recorded at 500 MHzH11F9iKD and RE values were determined against H11 scFvNDfND, not determined3.5ND95MonomerNDH11B2iKD and RE values were determined against H11 scFvNDfND, not determined2.0ND100MonomerNDa Expression yield per liter of bacterial cultureb GFC, gel filtration chromatographyc The solution properties of the various VH molecules are characterized as soluble (s) and structurally folded (f) with broad (b), sharp (s), and partially sharp (ps) NMR spectrad Folding was determined by one-dimensional 1H spectra recorded at 500 MHze Folding was confirmed by one-dimensional 1H and two-dimensional 1H-1H NOESY spectra recorded at 800 MHz. No concentration dependent line broadenings were observed from ∼30 to 300 μmf ND, not determinedg Sharp resonances at a concentration of 40 μm and partial line-broadening were observed at ∼1.0 mmh Folding was confirmed by one-dimensional 1H spectrum recorded at 800 MHzi KD and RE values were determined against H11 scFv Open table in a new tab The aggregation tendency of the human VHs was assessed in terms of their oligomerization state by gel filtration chromatography and NMR (TABLE TWO). All VHs were subjected to Superdex 75 gel filtration chromatography. Similar to a llama VHH, H11C7, all VHs gave a symmetric single peak at the elution volume expected for a monomer, and were essentially free of any aggregates (see the example for HVHP428 in Fig. 3A). In contrast, a typical human VH (i.e. BT32/A6) formed a considerable amount of aggregates. For three of the VHs, a minor peak with a mobility expected for a VH dimer was also observed. SPR analyses of the minor peaks gave off-rate values that were significantly slower than those for the monomer VHs, consistent with them being VH dimers. The dimer peak was also observed in the case of the llama VHH, H11C7. The folding and oligomerization states of the VHs at high concentrations were further studied by NMR spectroscopy. As shown in TABLE TWO, all the VH proteins studied appeared to be relatively soluble and assumed a well folded three-dimensional structure. One-dimensional NMR spectra of the VH fragments (Fig. 3B) showed structure folds characteristic of VH domains. The state of protein aggregation was also assessed by use of an PFG-NMR diffusion experiment for the HVHP414 fragment and two isoforms, VH14 and VH14-cMyc, with and without the c-Myc sequence, of the HVHP414. VH14 is a modified version of HVHP414 with a c-Myc N132E mutation and with an additional methionine residue at the N terminus. In brief, the PFG-NMR data (not shown) indicated that all the protein samples had expected monomeric molecular weights even at the relatively high protein concentrations used for NMR experiments.FIGURE 3Aggregation tendencies of the human VHs. A, gel filtration chromatograms comparing the oligomerization state of a human VH isolated in this study (HVHP428) to that of a llama VHH (H11C7) and a typical human VH (BT32/A6)