Consideration on Efficient Recombinant Protein Production: Focus on Substrate Protein-Specific Compatibility Patterns of Molecular Chaperones.

Expression of recombinant proteins requires at times the aid of molecular chaperones for efficient post-translational folding into functional structure. However, predicting the compatibility of a protein substrate with the right type of chaperone to produce functional proteins is a daunting issue. To study the difference in effects of chaperones on His-tagged recombinant proteins with different characteristics, we performed in vitro proteins expression using Escherichia coli overexpressed with several chaperone 'teams': Trigger Factor (TF), GroEL/GroES and DnaK/DnaJ/GrpE, alone or in combinations, with the aim to determine whether protein secondary structure can serve as predictor for chaperone success. Protein A, which has a helix dominant structure, showed the most efficient folding with GroES/EL or TF chaperones alone, whereas Protein B, which has less helix in the structure, showed a remarkable effect on the DnaK/J/GrpE system alone. This tendency was also seen with other recombinant proteins with particular properties. With the chaperons' assistance, both proteins were synthesized more efficiently in the culture at 22.5 °C for 20 h than at 37 °C for 3 h. These findings suggest a novel avenue to study compatibility of chaperones with substrate proteins and optimal culture conditions for producing functional proteins with a potential for predictive analysis of the success of chaperones based on the properties of the substrate protein.

Site-Specific Photocrosslinking to Immunoglobulin G Using Photoreactive Antibody-Binding Domains.

The high specificity and strong binding affinity of antibodies, most commonly immunoglobulin G (IgG), have led to their use in a wide range of research, diagnostic and therapeutic applications. Many of these applications require the antibody to be labeled with additional chemical or biological moieties. Here, we describe a method for the rapid and site-specific labeling of nearly any "off-the-shelf" IgG. Our method utilizes small photoreactive antibody-binding domains (pAbBDs) that are produced by modifying the IgG-binding domains of Protein A and Protein G with the unnatural amino acid benzoylphenylalanine (BPA). The pAbBDs are covalently linked to IgG heavy chains upon exposure to ultraviolet light. Fusion of pAbBDs to a given protein of interest or conjugation of pAbBDs with drugs, fluorophores, and/or other chemical moieties, enables the facile production of a diverse range of antibody conjugates.

A comprehensive review on staphylococcal protein A (SpA): Its production and applications.

The Staphylococcus aureus protein A (SpA) can be obtained through the culture of wild-type S. aureus and also as a recombinant protein in safe bacterial hosts. Several methods have been used to purify SpA among which ion-exchange chromatography, affinity chromatography, gel filtration, and per aqueous liquid chromatography (PALC) are common. SpA has a wide range of biochemical, biotechnological, and medical applications and is most commonly used in test methods such as immunoprecipitation, enzyme-linked immunosorbent assay, and Western blotting. SpA has also been widely utilized in pharmaceutical applications to bind to immune complexes and serum immunoglobulins. SpA also directly binds to the B-cells preventing initiation of infectious diseases as well as having a role in the development of various autoimmune diseases. This review considers different applications of SpA in biotechnology and its novel clinical application for effective treatment of autoimmune diseases. It also discusses various strategies for expression and purification of the SpA including types of column chromatography that are commonly used in protein purification and developing SpA surface display technologies. Finally, this review highlights the potential and novel applications of SpA immobilization, SpA typing, protein engineering for further development of immunological and biochemical research, and also application of SpA as a diagnostic biosensor.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application.

Aequorin is a Ca2+-binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL.

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms.

A novel benzimidazole molecule that was identified in a small-molecule screen and is known as antibiofilm compound 1 (ABC-1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 µM ABC-1 was tested in various biofilm-forming strains of S. aureus. It was demonstrated that ABC-1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall-associated protein dependent or cell wall- associated extracellular DNA (eDNA). Of note, ABC-1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC-1 treated strains, implying that ABC-1 affects not only SpA but also other factors. Indeed, ABC-1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC-1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.

Protein A is released into the Staphylococcus aureus culture supernatant with an unprocessed sorting signal.

The immunoglobulin binding protein A (SpA) of Staphylococcus aureus is synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion.

Community-acquired meticillin-resistant Staphylococcus aureus strain USA300 resists staphylococcal protein A modulation by antibiotics and antimicrobial peptides.

Community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) causes severe diseases through virulence factors such as staphylococcal protein A (SpA), which favours immune evasion. We have previously shown that antimicrobial peptides (AMPs) and antibiotics decrease SpA expression in CA-MRSA strains. Here we examined the effects of antibiotics and AMPs, alone and in combination, on SpA expression in various CA-MRSA strains. Six S. aureus isolates corresponding to the major worldwide CA-MRSA clones (ST8-USA300, ST80 and ST30) were selected. Strains were cultured to exponential growth phase and were subsequently incubated with antibiotics (tigecycline, linezolid, clindamycin and vancomycin) at 0.25× MIC or with AMPs [human neutrophil peptide (HNP)-1-3] at the LD50, alone and in combination. After 6h, cultures were assessed for spa mRNA by RT-PCR, whilst SpA protein was measured by specific ELISA after 18h. When used alone, antibiotics (clindamycin, linezolid and tigecycline) or HNPs significantly reduced both SpA production and mRNA levels in ST30 and ST80 strains. When used in combination, HNPs and clindamycin, linezolid or tigecycline synergistically reduced SpA production (6-100-fold) and spa mRNA levels (4-20-fold) in ST80 and ST30 strains. In contrast, for USA300 strains, among all antibiotics, clindamycin alone reduced SpA production (3.5-fold), whereas with combined treatments including HNPs, only a slight reduction in SpA production (1.7-2.2-fold) was observed. In conclusion, antibiotics and AMPs do not modulate SpA expression in USA300, unlike in other CA-MRSA clones. This observation suggests that the virulence and successful spread of USA300 strains is associated with a specific regulatory network.

In vitro molecular evolution yields an NEIBM with a potential novel IgG binding property.

Staphylococcus aureus protein A (SpA) and protein G of groups C and G streptococci (SpG) are two well-defined bacterial immunoglobulin (Ig)-binding proteins (IBPs) with high affinity for specific sites on IgG from mammalian hosts. Both SpA and SpG contain several highly-homologous IgG-binding domains, each of which possesses similar binding characteristic of the whole corresponding proteins. Whether specific combinations of these domains could generate a molecule with novel IgG-binding properties remained unknown. We constructed a combinatorial phage library displaying randomly-rearranged A, B, C, D and E domains of SpA as well as the B2 (G2) and B3 (G3) domains of SpG. In vitro molecular evolution directed by human, rabbit, bovine, or goat polyclonal IgGs and four subclasses of mouse monoclonal IgGs generated one common combination, D-C-G3. A series of assays demonstrated that D-C-G3 exhibited a potential novel IgG binding property that was obviously different from those of both parent proteins. This study provides an example of successful protein engineering through in vitro molecular evolution and useful approaches for structure and function studies of IBPs.

Genome-wide analysis of the regulatory function mediated by the small regulatory psm-mec RNA of methicillin-resistant Staphylococcus aureus.

Several methicillin resistance (SCCmec) clusters characteristic of hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains harbor the psm-mec locus. In addition to encoding the cytolysin, phenol-soluble modulin (PSM)-mec, this locus has been attributed gene regulatory functions. Here we employed genome-wide transcriptional profiling to define the regulatory function of the psm-mec locus. The immune evasion factor protein A emerged as the primary conserved and strongly regulated target of psm-mec, an effect we show is mediated by the psm-mec RNA. Furthermore, the psm-mec locus exerted regulatory effects that were more moderate in extent. For example, expression of PSM-mec limited expression of mecA, thereby decreasing methicillin resistance. Our study shows that the psm-mec locus has a rare dual regulatory RNA and encoded cytolysin function. Furthermore, our findings reveal a specific mechanism underscoring the recently emerging concept that S. aureus strains balance pronounced virulence and high expression of antibiotic resistance.

Optimization of extracellular truncated staphylococcal protein A expression in Escherichia coli BL21 (DE3).

Staphylococcal protein A (SpA) plays an important role in Staphylococcus aureus pathogenesis. The recombinant SpA is also widely used in biotechnology to purify polyclonal and monoclonal immunoglobulin G antibodies. In this study, expression and secretion of a truncated form of SpA containing five immunoglobulin-binding domains using its own native signal sequence were optimized in Escherichia coli. Optimization was carried out using response surface method (RSM), making use of the interaction between five variables. The initial results revealed that the signal peptide from S. aureus was recognized in E. coli and the resulting SpA was expressed and secreted into the medium. Compounds, such as glycine, affected the secretion of SpA into the culture medium. The central composite design experiment showed that the optimum conditions for the maximum expression of recombinant truncated SpA in E. coli included 10% (w/v) lactose, 1.77% (w/v) glycine, induction time of 11 H, an optical density (600) of 1.1, and a temperature of 33 °C. Optimization using RSM resulted in a fivefold increase in the secretion of SpA. To date, this is the first study of its kind regarding the definite influence of glycine concentration and duration of the cultivation period on the secretion of SpA.

Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein.

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.

Effects of subinhibitory concentrations of antibiotics on virulence factor expression by community-acquired methicillin-resistant Staphylococcus aureus.

OBJECTIVES: To examine the effect of subinhibitory concentrations (sub-MICs) of antistaphylococcal drugs on Panton-Valentine leucocidin (PVL), α-haemolysin (Hla) and protein A (SpA) expression by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). METHODS: Five clinical isolates representing the main worldwide CA-MRSA clones were grown with sub-MICs (1/8, 1/4 and 1/2 MIC) of five antibiotics (clindamycin, daptomycin, linezolid, tigecycline and vancomycin). After 4 and 6 h of incubation, culture pellets were used for relative quantitative RT-PCR with primers specific for pvl, hla, spa and gyrB. The PVL, Hla and SpA concentrations were measured in the supernatant (for PVL and Hla) and in the cell pellet (for SpA) using specific ELISAs. RESULTS: For all strains tested, clindamycin and linezolid dramatically reduced mRNA levels of PVL and SpA. Tigecycline also decreased the PVL and SpA mRNA levels of 3/5 and 4/5 strains tested, respectively, whereas daptomycin and vancomycin had no significant effect. PVL and SpA quantification confirmed the concentration-dependent inhibition of PVL and SpA production by clindamycin and, to a lesser extent, by linezolid and tigecycline. Only clindamycin decreased Hla mRNA expression, whereas linezolid, tigecycline and daptomycin showed heterogeneous strain-dependent results, and vancomycin had no significant effect. Analysis of the Hla level revealed a stronger concentration-dependent inhibition of Hla release by clindamycin than by linezolid. CONCLUSIONS: The effect of sub-MICs on virulence expression depended on the antibiotic and the virulence factor. Clindamycin and linezolid consistently suppressed the expression of different virulence factors by CA-MRSA, whereas tigecycline specifically suppressed PVL expression. Daptomycin and vancomycin seem to have no significant effects at these concentrations.

Functional variation reflects intra-strain diversity of Staphylococcus aureus small colony variants in the host-pathogen interaction.

It is well acknowledged that genetic variation accounts for the intra-species variability in Staphylococcus aureus isolates. Similarly, deficiency in DNA repair and the resulting increase in genomic mutations determine intra-strain variability in S. aureus small colony variants (SCV). The aim of this study was to investigate whether intra-strain diversity would be associated with an alteration of the host-pathogen interaction. To this end, biofilm formation and immune stimulatory capacity were compared in consecutive SCV isolates originating from a single patient. Despite the relatedness of the isolates, the results revealed significant differences in biofilm formation and immune stimulation determined by Toll-like receptor-2 (TLR2) activity. Variation in the extent of biofilm production could be attributed to differences in the expression of protein A (SpA) and agrA. TLR2 activity only partially correlated with these parameters. Although transiently increased functional activity correlated with clinical remission and was abrogated in MRSA superinfection, we can only speculate that changes in the SCV phenotype reflect alterations in the microbial environment and/or treatment. Taken together, our study provides in vivo evidence for the functional consequences of intra-strain variation in S. aureus.

[Preparation and characterization of recombinant protein A affinity packing].

To obtain an excellent antibody purification medium, affinity chromatographic packing with recombinant staphylococcal protein A (rProtein A) was synthesized and verified. With E. coli cells harboring the recombinant plasmid, the rProtein A was expressed and purified, then was conjugated to epichlorohydrin-activated Sepharose 4 Fast Flow to prepare an affinity chromatographic packing. The performances of the packing were validated with rabbit antiurate oxidase. After the reaction, the concentration of rProtein A coupled to Sepharose 4 Fast Flow was 1.5 x 10(-4) mol/L. Scatchard analysis of the binding isotherm for IgG showed excellent binding capacity on the adsorbent, giving a dissociation constant (Kd) of 2.28 x 10(-7) mol/L and a theoretical maximum adsorption capacity of 20. 697 g/L. The identification showed the packing was stable in 0.1 mol/L NaOH solution at 1 h. By using the packing, the pure antibody exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from rabbit serum after one-step elution, with 96. 1% of yield and 19 mg IgG for one milliliter of gel. The research laid the foundation of the localization of rProtein A affinity packing.

Large-scale transfection of mammalian cells.

The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins (r-proteins) to be obtained for fundamental or clinical research. In this article, we describe a one-liter transfection using polyethyleneimine (PEI) for DNA delivery into human embryonic kidney (HEK-293) cells cultivated in serum-free suspension to produce a recombinant human monoclonal antibody that yields up to about 1 g/L in a 10-day process. The method is based on a DNA delivery step performed at high cell density (20×10(6) cells/mL) by direct addition of DNA and PEI to the culture. Subsequently, the cells are diluted 20-fold for the 10-day production phase in the presence of valproic acid (VPA), a histone deacetylase inhibitor. The methods for plasmid purification, antibody quantification by enzyme-linked immunosorbent assay (ELISA), and affinity purification with protein A are also described.

[Cloning, expression and screening tandem repeats of the Z domain of Staphylococcus aureus protein A].

To screen an efficient recombinant Staphylococcus aureus protein A (SpA) for preparing matrix for affinity purification of immunoglobulin G (IgG), a genetic engineering approach was used to obtain monomer, two, three, four and five tandem repeats genes of the Z domain of SpA, then the genes were cloned into expression vector pET-22b and subsequently expressed in Escherichia coli BL21 (DE3). After induction with lactose, the target proteins were purified by Ni2+ affinity chromatography. The proteins with two, three, four and five tandem repeats of the Z domain were then coupled to CNBr-activated Sepharose 4B as an affinity chromatography matrix for affinity purification of human IgG. Furthermore, the differences in protein yield and IgG-binding capacity at different recombinant proteins were analyzed. The target proteins with monomer and tandem repeats of the Z domain had an effective expression in the genetic engineering bacteria. IgG could be specifically absorbed from human plasma by affinity chromatography. The protein yield and amount of IgG absorption of per mole protein could be improved by increasing the tandem repeats number of the Z domain. Compared with other tandem repeats, four tandem repeats of the Z domain exhibited more protein yield (160 mg/10 g wet cells) and higher level of IgG absorption (34.4 mg human IgG/mL gel). Therefore, four tandem repeats of the Z domain is more suitable for preparing matrix for affinity purification of IgG.

Modulation of virulence gene expression by cell wall active antibiotics in Staphylococcus aureus.

OBJECTIVES: To investigate the effect of subinhibitory concentrations of cell wall active antibiotics on virulence gene expression and biofilm formation in Staphylococcus aureus Newman and in laboratory strains. METHODS: Promoter regions of spa, lukE and agr RNAIII were cloned upstream of a modified luxABCDE reporter. Using disc diffusion assays, the effects of antibiotics were observed on gene expression and quantitative real-time PCR was employed to confirm the results. Assays were performed to measure biofilm formation in wild-type S. aureus and respective spa-deficient and small colony variant mutants in the presence of subinhibitory concentrations of antibiotics. RESULTS: Expression of spa and lukE was stimulated by subinhibitory concentrations of penicillin and cefalotin, while agr RNAIII expression was not affected. Denser biofilms were formed by S. aureus Newman and its small colony variant in the presence of subinhibitory concentrations of cefalotin. CONCLUSIONS: Subinhibitory concentrations of certain antibiotics have been shown to stimulate virulence gene expression in S. aureus; this may alter the progression of infection and thus render antimicrobial therapy unreliable. The use of appropriate combinations of antibiotics might be an approach to avoiding this situation. Promoter-lux reporters are sensitive tools for studying the modulation of transcription by antibiotic inhibitors, and could be used to predict novel therapeutic combinations for the treatment of infection.

TNF receptor I on human keratinocytes is a binding partner for staphylococcal protein A resulting in the activation of NF kappa B, AP-1, and downstream gene transcription.

Primary human keratinocytes and immortalized HaCaT cells were analysed for their capacity to bind purified staphylococcal protein A (SpA). Co-incubation with FITC-labelled SpA led to a dose-depending attachment. Pull-down experiments with cellular extracts revealed the TNFα receptor I (TNF RI) as binding partner on keratinocytes. Thus, we next looked for expression of this receptor in human epidermis and cultured keratinocytes. TNF RI is strongly expressed on all keratinocytes analysed, both at the mRNA and protein level and activation by SpA at optimal doses of 50-100 µg/ml resulted in the phosphorylation of the TNF RI downstream kinases MEK1/2, JNK1/2, and p38 subsequently leading to translocation of the p65 NF kappa B subunit and AP-1 into the nucleus. This translocation was then followed by increased expression of IL-8 and COX-2, two known NF kappa B-induced pro-inflammatory genes. To further test the relevance of our findings, we analysed in vitro production of over 100 strains isolated from atopic eczema showing that more than 85% of the tested strains produced extracellular SpA in substantial amounts. Thus, besides superantigens, haemolysins, and other cell wall components, Staphylococcus aureus exerts pro-inflammatory stimuli on human keratinocytes through the production of SpA signalling through TNF RI.

Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C-staphylococcus A protein.

Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC-glial cell line-derived neurotrophic factor or gC-brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC-ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2 months) expression.