Commercial Matrixs of Protien G

Protein-G Sepharose 4 Fast Flow: is manufactured by GE Healthcare (Hober, J. Chromatogr B, 2007, 848, pp. 40-47 and contains SpG immobilized as a ligand. 

Introduction

Protein G is a cell surface protein from Streptococcus: it contains multiple copies of two different small domains which can independenlty bind albumin and IgG. It is widely used as an affinity ligand for the purificaiton of IgG. (Palmer, J. Biotechnology 134 (2008) 222-230)

Compared to SPA, SPG displays a different binding sepctra for immunoglobulins from different species and subclasses thereof. The IgG binding domains of protein G are now widely used as an immunological tool for the affinity purification of monoclonal antibodies. Production of subfragments constructued by DNA technology have shown that an individaul C region is sufficient for full IgG binding. Nilsson (US6,740,734). 

Generally, protein G binds more IgG subclasses and with higher affinity than protein A. Protein G binds to human IgG of all four subclasses as well as to a number of mammalian monoclonal antibodies including those form mosue and rate, whereas protein A binds to neitehr human IgG3 nor to rat IgG. All IgGs that have been examined show a higher bnding affinity to protin G than to protin A. Protein G also interacts weakly with Fab, the antigen-bnding fragment of IgG but its binding affinity is only aobut 10% of the affinity for Fc. (Jones “Crystal structure of the C2 fragmetn of streptococcal protein G in complex with the Fc domain of human IgG” Structure, 1995, 3: 265-278). 

Protein G is a good choice for general purpose capture of antibodies at laboratory scale since it binds a broader range of IgG from eukaryotic species and binds more classes of IgG than protein A. Usually, protein G has greater affinity for IgG than protein A and exhibits minimal binding to albumin, resulting in cleaner preparations and greater yields. (GE Healthcare “Affinity Chromatography”, copyright 2014-2016) 

Unpredictability of IgG binding

Hober (Protein Engineering 15(1) pp. 835-842, 2002) discloses that while single mutations N7A and N36A in the C domain of SpG resulted in constructs with retained affinity to Fc, a significant decrease in affinity to IgG was observed when substituting Asn34.  see also Kossiakoff, (J of Immunological Methods, 415, pp. 24-30, 2014, citing Hober and stating the N7A/N36A double mutant led to a loss of Fab bnding affinity, presumably because of the critical contacts Asn36 provide in the Protein G-Fa complex.); see also US 2018/0044385)

Kossiakoff, (J of Immunological Methods, 415, pp. 24-30, 2014); see also US 2018/0044385) discloses that an 8 point mutations of the C2 domain of Protein G demonstrated a preference for the human IgG1 isotype as its affinity was decreased by a factor of 20-50 fold for IgG2, IgG3 and IgG4, suggesting subtile differences in domain orientation impact binding. 

Structure of Protein G

Streptococcal protein G cinludes two or three domains that bind to the constant Fc region of most mammlain IgGs. Protein G is functionally related to staplylococcal protein A, with which it shares neither sequence nor structural homlogy. Sauer-Erikson (Structure, 1995, 3(3), 265-278). 

The beta-1, beta-2 and beta-3 domains of SpG carry out molecular recognition of both the Fc part of IgG and the Fab part of the homo sapiens IgG. The amino acid sequene of these domains differ by originating bacteria kinda and strains. Some subspecies lack beta-3 domain. (Koneka Corp, Japanese Patent Publication number 2009-195184)

B1 domain: is a 56 residue domain that foled into a four-stranded D-sheet and one a-helix. Despite its small size, it has two separate IgG binding sites on its surface, each interacting respectively with specific, independent istes on the Fab or Fc fragments of the antibody. Hellinga (US 6,663,862). 

C1,C2 C3 domains: Streptococcal protein G is a mltidomain protein with seaprate albumin binding and immunoglobulin binding regions. SPG exhibits a braod spectrum of binding to IgG subclasses, and bind to all four human subclasses of IgG as well as several animal IgG subclasses. The IgG binding region consists of three domains, C1, C2 and C3, exhibiting high sequence homology. The C2 domain is a small protein composed of only 55 residues. Hober (Protein Engineering 15(1) pp. 835-842, 2002)

The C2 domain of Protein G from Streptococcus is a multi-specific prtoein domain; it possess a high affinity for the Fc region of IgG but a much lower afifinity for the constant domain of the antibody fragment Fab. (Kossiakoff, J of Immunological Methods, 415, pp. 24-30, 2014); see also US 2018/0044385). 

there are only six differences in the amino acid sequences of the C1 and C3 domains of protien G. Nevertheless, the C3 domain binds IgG about seven times tighter than the C1 domain. (Jones “Crystal structure of the C2 fragmetn of streptococcal protein G in complex with the Fc domain of human IgG” Structure, 1995, 3: 265-278).

Where/What Protein G binds

It has been shown that binding to intact IgG as well as antibody fragments such as F(ab’)2 and Fc regions by protein G. Protein G also binds to immunoglobulins of most species including rat and goat and recognises most classes and subclasses. (Darcy , chapt 20 from Protein Chromatography: Methods and Protocols, Methods in Molecular Biology, vol. 681 (2011).

IgG binding:

Protein G recognizes a common site at the interface between CH2 and CH3 domains on the Fc part of human IgGA1, IgG2, IgG3 and IgG4 antibodies (Fcgamma) with high affinity. In addition, Protein G shows binding to the Fab portion of IgG antibodies through binding to the CH1 domain of IgG in combination with a CL domain of the kappa isotype. Protein G only binds to Fab from IgG1, IgG3 and IgG4 but not to Fab of IgG2. Binding affinity towards CH1 is significantly lower compared to its epitope on the Fc part.  (Hermans US13/982970). 

Protein G and protein A have developed different starties for binding to Fc. The protein G-Fc complex involves mainly charged and polar contacts, whereas protein A and Fc are held together through non-specific hydrophobic interactions and a few polar interactions. Erikson (Structure, 1995, 3(3), 265-278)

Protein G binds Fc at the hinge region that connects the CH2 and CH3 domains. There are three residues of the CH2 domain of Fc which are involved in the interfacial interacts: Ile253, Ser254 and Gln 311. In the CH3 domain, there are two areas which contribute to the interface: Glu380 and Glu382 and the residues His433-Gln438. All residues that interact with protein G are situated within loop regions of Fc except for Glu380, Glu382 and Gln438 which are exposed on one of the beta-hseets making up the CH3 domain. (Jones “Crystal structure of the C2 fragmetn of streptococcal protein G in complex with the Fc domain of human IgG” Structure, 1995, 3: 265-278).

Serum Albumin binding:

Streptococcal protein G (SpG) is capable of binding to both IgG and serum albumin. The structure of one of the three serum albumin binding domains has been deteremind, showing a three helix bundle domain, named ABD (ablumin binding domain) and is 46 amino acid residues in size. It has also been designated G148-Ga3. (Abrahmsen, WO/2009/016043)

Conditions used:

Proudfoot (Protein Expression and Purificaiton 8, 368-373 (1992) disclsoes purification of a Fab’ fragment by protein G sepharose. After the cell culture sueprnatant was applied to the column a peak of absorbance at 280 nm of weakly bound material was recoverd with a pH 7 wash (peak A). A further peak (peak B) could then be eluted from teh column with a low pH wash. Binding to teh protein G-Sepharose was efficient with no Fab’ ro F(ab’)2 deteced in the column flow through. 

Analogues of Protein G: Beta1/C2 domain mutants:

An SpG functional domain having an IgG binding ability is referred to as the beta domain or C domain. (Yoshida, US 16/585,895, published as US 2020/0031915)

The C2 domain of Protein G from Streptococcus is a multi-specific protein domain; it possesses a high affinity for the Fc region of the IgG, but a much lower affinity for the constant domain of the antibody fragment (Fab), which limits some of its applications. Accordingly, the Protein G interface has been engineered using phage display to create a Protein G variant with beta point mtuations to provide about 100 fold improved affinity over the parent domain for Fab fragments. A variant was also isolated having enhanced stability to basic conditions whcih is useful for regeneration. In some embodiments, the protin G Fab binding region is modified to have Tyr at position 16, Gln, His, or Tyr at position 452 or Tyr or Phe at position 40. Also disclsoed is pruificaiton of Fabs using the variants with a pH dependence of elution.  (See Kossiakoff (US 2018/0044385 and Baily, J of Immunoglobical Methods, 415 (2014) 24-30).

Deletions at N/C terminus:

The N-terminal of wild SpG-beta1 is Asp but it is Thr in wild type SpG-B1. The N-terminal is not contained in (Hober, Protein Engineering, 15(1), pp. 835-842, 2002) and the kind of amino acid at the N-temrinal makes no difference. (Yoshida, US 16/585,895, published as US 2020/0031915). 

Improved Alkali stability:

–Replacement of Asn residues: 

Protein G is a cell wall protein form group C and G streptococci. It is a type III Fc receptor which binds with high affinity to the Fc region of antibodies, in particular, IgG antibodies. It has been shown that Asn residues are the most susceptible at extreme alkaline pH and that replacement of all 3 Asn residues within the IgG binding domain of PrtG improves stability towards caustic alkali by about 8 fold (Palmer, J. Biotechnology 134 (2008) 222-230).

—-Substitution at Position 8:

Hober (Protein Engineering 15(1) pp. 835-842, 2002) disclsoes that the C2 domain contains three asparagine residues which were substituted for alanines since no alternative amino acids were found in the homologous sequences. Three single mutants were designated C2 N7A, C2 N34A and C2 N36A. 

Lindman (Biophysical J. 90, 2006, pp. 2911-2921) teaches a variant of protien G B1 domain having 3 mutations which include N8D. 

Palmer, (J. Biotechnology 134 (2008) 222-230) discloses that Asp8 lies within a beta-strand and that a N8T/N35A double mutant showed 17 fold more stability. 

Yoshida (US 16/585,895, published as US 2020/0031915) discloses an altered Protein G which can bind to an Fc region and a Fab region of an immunoglobulin and which has excellent chemical stability to an alkaline solution which includes for example the beta-1 region of SpG where the 8th position is substituted by Asp, Glu, His, Ile, Lys, Leu or Val. 

Mutants having higher binding activity for the Fab region (useful for purificaiton of Fab fragments):  

Since SpG has a weak binding affinity to a Fab region, the performance of a SpG affinity separation matrix product to maintain an antibody fragment which does not contain a Fc region and which contains a Fab region only is considered to be insufficient. Accordingly, efforts ahve been made to improve the binding affinity of SpG to a Fab region by introducing a mutation into SpG. (Murata and Yoshida, US 2017/03349457). 

—-27Glu, K28, W43, Y45:

Hellinga (US 6,663,862 and WO 00/74728) discloses B1 Protein G mutants which exhibit binding activity for a Fab fragment of an IgG but substantially no binding activity for a Fc fragment of an IgG such as one having a mutation at the glutamate 27, lysine 28, tryptophan 43 and tyrosine 45. 

—-13T/S, 19V/L/I, 30V/L/I, 33F:

Yoshida (US 14/914439, published as US2016/0289306; see also Murata and Yoshida, US 2017/03349457) discloses a B1 domain of protein G with substitution of an amino acid residue at not less than the 13th, 19th, 30th and 33rd positions and having a higher binding force to the Fab region .  Specifically, a humanized monoclonal IgG was fragmented into a Fab fragment and a Fc fragment using pepain, and only the Fab fragment was seaprated and purified. 

(Koneka Corp, Japanese Patent Publication number 2009-195184) discloses variant protein G beta domains which bind more strongly to IgG-Fab fragment. 

Multimers of Protein G

Kossiakoff (Kossiakoff, J of Immunological Methods, 415, pp. 24-30, 2014); see also US 2018/0044385) discloses a fusion between to or mroe polypeptides of protein G variants. Multi-valency is a common feature of many biological systems that ahrness the simultaneous engagement of tethered ligands or multiple receptors. Biological processes use this as a means to increase the effective affinity of weak binding ligands as well as to qulitatively modify the activity of proteins through a multi-valent engagemnt and molecular crosslinking. Antibodies exploit multi-valency thorugh natrually occruing formats including the IgG (bivalent), IgA (tetravelnt) and IgM (decavalent).