December 2000
Volume 41, Issue 13
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Glaucoma  |   December 2000
Recombinant TIGR/MYOC Increases Outflow Resistance in the Human Anterior Segment
Author Affiliations
  • Michael P. Fautsch
    From the Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
  • Cindy K. Bahler
    From the Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
  • David J. Jewison
    From the Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
  • Douglas H. Johnson
    From the Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
Investigative Ophthalmology & Visual Science December 2000, Vol.41, 4163-4168. doi:
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      Michael P. Fautsch, Cindy K. Bahler, David J. Jewison, Douglas H. Johnson; Recombinant TIGR/MYOC Increases Outflow Resistance in the Human Anterior Segment. Invest. Ophthalmol. Vis. Sci. 2000;41(13):4163-4168.

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Abstract

purpose. To determine the effect of human recombinant TIGR/myocilin (MYOC) protein on outflow resistance in the human anterior segment.

methods. A cDNA for MYOC was inserted into a bacterial expression system and purified with nickel ion affinity chromatography. The anterior segments of 12 pairs of human eyes were placed in perfusion organ culture. One eye received an anterior chamber exchange with partially purified recombinant MYOC (25 μg), whereas the other eye received either heat-denatured recombinant MYOC (25 μg), partially purifiedβ -galactosidase (25 or 250 μg), or partially purified control proteins isolated from a null expression lysate (25 μg). Eyes were fixed up to 72 hours after infusion, and immunohistochemistry was performed using anti-MYOC polyclonal antibody.

results. Recombinant MYOC caused an increase in IOP over 12 hours, increasing outflow resistance 94%, whereas the fellow eye infused with null expression sample increased 12% (n = 7; P = 0.0005). When compared with recombinant MYOC, neither heat-denatured MYOC, recombinant β-galactosidase, bovine serum albumin, nor fetal calf serum caused an increase in outflow resistance. MYOC IOP remained above baseline levels for 48 to 72 hours. Immunohistochemistry results confirmed the presence of recombinant MYOC in the trabecular meshwork.

conclusions. Recombinant MYOC increased outflow resistance in human anterior segments, whereas control proteins did not. MYOC may increase outflow resistance by specific interactions within the trabecular meshwork.

In 1997, Stone and colleagues 1 reported that mutations in the trabecular meshwork–inducible glucocorticoid response (TIGR) gene were present in patients with JOAG and POAG. TIGR was originally identified as a secreted protein that was induced in trabecular cells after prolonged treatment with glucocorticoids. 2 Independent of the TIGR studies, a novel cytoskeletal protein was identified in the retina and named myocilin. 3 Subsequent database searches revealed that the genes producing the TIGR and myocilin proteins were the same, and this gene has been designated MYOC by the HUGO Nomenclature committee. 
MYOC mRNA is present in a variety of ocular and nonocular tissues. 3 4 5 6 7 8 9 It is induced by stress, such as H2O2 and mechanical stretch, and steroid treatment. 2 10 11 In the trabecular meshwork, in situ hybridization studies reveal the presence of MYOC mRNA within the uveal, corneoscleral, and juxtacanalicular regions but only occasionally in the endothelial cells of Schlemm’s canal. 12 MYOC protein is found on trabecular cells throughout meshwork, and also in the extracellular material in the juxtacanalicular region. 13 In glaucomatous eyes, MYOC can be more abundant than in normal eyes and is also found throughout the trabecular meshwork. 14  
The function of MYOC protein is unknown. MYOC is present in aqueous humor and is secreted by human trabecular meshwork cells in both monolayer and perfusion organ culture after treatment with glucocorticoids. 15 16 Consistent with this, MYOC has an N-terminal 32 amino acid signal peptide sequence, which is usually found in secreted proteins. 17 MYOC also has a potential intracellular role in the photoreceptors of the retina and has also been shown to colocalize with microtubules in transfection studies. 3 18  
To investigate the role of MYOC in aqueous outflow, we made recombinant MYOC and report that infusion of recombinant MYOC into human anterior segments of the eye increases intraocular pressure. 
Materials and Methods
Plasmid Construction
A 1250-bp MYOC cDNA was isolated by RT-PCR using total RNA from human trabecular meshwork. 12 Additions to the 5′ and 3′ end of the 1250-bp MYOC cDNA by several rounds of PCR resulted in a 1418-bp fragment (FAD1: 5′-ACCAGAGTGGCCGATGCCAGTATACCTTCAGTGTGGCCAGTCCCAATGAATCCAG-3′; FAD2: 5′-GGACAGCTCAGCTCAGGAAGGCCAATGACCAGAGTGGCCGATGCCAG-3′; RAD1: 5′-GGGGGTTGT-AGTCAATCATGCTGCTGTACTTATAGCGGTTCTTGAATGGGATGGTC-3′; RAD2: 5′-GTTCAAGTTGTCCCAGGCAAAGAGCTTCTTCTCCAGGGGGTTGTAGTCAATCATGC-3′; RAD3: 5′-TCACATCTTGGAGAGCTTGATGTCATAAGTGACCATGTTCAAGTTGTCCCAGGCAAAGAGC-3′; BacEx5: 5′-ATGCGGATCCAAGGACAGCTCAGCTCAGGAAGGCC-3′; BacEx3: 5′-ATGCAAGCTTATCACATCTTGGAGAGCTTGATGTC-3′). This fragment contained a 5′-BamHI and 3′-HindIII restriction enzyme cleavage site in addition to the full coding region of MYOC minus the signal peptide sequence (133–1551 bp). Digestion of the 1418-bp fragment with restriction enzymes BamHI and HindIII resulted in a fragment that was cloned into the BamHI/HindIII restriction sites in the bacterial expression plasmid pRSET-B (Invitrogen, Carlsbad, CA). The resulting plasmid was called pRSET-MYOC. Sequence of pRSET-MYOC was verified in both directions using a Perkin Elmer/Applied Biosystems (Foster City, CA) DNA sequencer. 
Protein Purification
The expressed product from the pRSET-MYOC plasmid produces a fusion protein that adds 32 amino acids to the N terminus of MYOC (Note: This replaced the 32 amino acid signal peptide). Within the 32 amino acids are a 6 amino acid histidine tag (for purification) and an 8 amino acid epitope that is recognized by monoclonal antibody anti-Xpress (Invitrogen). 
Plasmid pRSET-MYOC, pRSET-β-galactosidase or pRSET-B (empty expression vector serving as negative control for purification) were transformed into BL21(DE3)pLyS bacteria. Overnight cultures were diluted to 0.1 (OD600) and allowed to grow until OD600 reading was 0.4 to 0.5 (exponential growth). The culture was allowed to grow for 3 additional hours, and then cells were collected by centrifugation. For purification of MYOC,β -galactosidase, and control proteins (pRSET-B), bacterial pellets were lysed in 50 mM sodium phosphate buffer (per liter: 46.6 ml of 1 M Na2HPO4, pH to 8.0 with 1 M NaH2PO4), 300 mM NaCl, 10 mM imidizole, and 1 mg/ml lysozyme. Bacterial lysate was sonicated, pulled through a 23-gauge needle, and centrifuged to remove debris. Supernatant was collected and 1.5 ml of Ni-NTA agarose (Qiagen, Valencia, CA) was added. The sample was allowed to rock at 4°C for 1 to 2 hours and centrifuged, and the supernatant was discarded. Ni-NTA agarose was washed six times with 10 ml of 50 mM sodium phosphate buffer, 300 mM NaCl, and 20 mM imidizole. Two additional washes were performed with 50 mM sodium phosphate buffer, 300 mM NaCl, and 50 mM imidizole. Protein was eluted from Ni-NTA agarose with 1 to 2 ml of 50 mM sodium phosphate buffer, 300 mM NaCl, and 250 mM imidizole. Isolated protein was dialyzed overnight against buffer containing 50 mM sodium phosphate buffer, 300 mM NaCl, and decreasing amounts of imidizole (100 mM → 50 mM → 0 mM). 
TIGR Antibody
A 25 amino acid peptide homologous to MYOC amino acids 108 to 131 was made on a Perkin Elmer/Applied Biosystems Peptide Synthesizer and sent to Cocalico Biologicals (Reamstown, PA) for antibody production in rabbits (peptide sequence: ARPQETQEGLQRELGTLRRERDQLC). The resulting anti-MYOC antibody was purified by running the rabbit serum over a protein G column to purify the IgG fraction. Purified anti-MYOC IgG was further purified by bacterial lysate affinity chromatography (Pierce, Rockford, IL). 
Western Blot Analysis
Various samples from the bacterial purification of recombinant MYOC were collected and boiled for 3 minutes in 125 mM Tris (pH 6.8), 20% glycerol, 4.6% SDS, 10% β-mercaptoethanol, 0.001% bromophenol blue. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane in 49.6 mM Tris, 384 mM glycine, and 0.01% SDS. After blocking the membrane in 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween, and 2% evaporated milk, mouse monoclonal anti-Xpress or rabbit polyclonal anti-MYOC antibody was added. Transfers were washed three times with 20 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween. Horseradish peroxidase–linked anti-mouse Ig (Amersham, Piscataway, NJ) or anti-rabbit Ig (Amersham) was used as secondary antibodies. Antibody:antigen complex was detected using ECL Western blotting signal detection reagent (Amersham). 
Human Organ Cultures
Normal human eyes were obtained at autopsy within 20 hours of death and placed in anterior segment perfusion culture. The average age of the donor eyes was 71 ± 8 years (range, 58–80 years). No eyes had glaucoma or uveitis or had been treated with topical medications. The culture technique was similar to that described previously. 19 20 21 Eyes were bisected at the equator, and the iris, lens, and vitreous were removed. The anterior segment was clamped in a modified Petri dish, and the eye was perfused with Dulbecco’s modified Eagle’s medium (Cat. no. 10-017-CV; Cellgro, Herndon, VA) with added antibiotics (penicillin, 10,000 units; streptomycin, 10 mg; amphotericin B, 25 mg; and gentamicin, 1.7 mg in 100 ml medium) at the normal human flow rate (2.5 μl/min). The anterior segments were cultured at 37°C in a 5% CO2 atmosphere. Intraocular pressures were continuously monitored with a pressure transducer connected to the second access cannula built into the dish and recorded with an automated computerized system. 
After an initial adaptation period in culture (2–7 days), one anterior segment of a pair was given partially purified recombinant MYOC (25μ g diluted to 900 μl in phosphate-buffered saline [PBS]) via an anterior chamber exchange. The fellow control eye was given control proteins purified from pRSET-B lysates (25 μg diluted to 900 μl in PBS) also via anterior chamber exchange. The anterior chamber exchanges were performed by using a gravity-driven, constant pressure method over a 10-minute period. Pressure data from either eye were not used until the end of the first hour after the anterior chamber exchange. 
Several control protein samples were also tested in the organ perfusion culture system. The following samples and amounts were tested: heat-denatured recombinant MYOC (25 μg boiled for 10 minutes), partially purified β-galactosidase (25 or 250 μg), bovine serum albumin (250 μg), fetal bovine serum (25, 250, 1000, 2500, or 25,000μ g), and human serum (25, 250, 1000, 2500, or 65,000 μg). All controls were diluted to 900 μl with PBS where appropriate and delivered to eye by anterior chamber exchange. 
Statistical Analysis
Outflow resistance was calculated as R = P/F, and data are expressed as a percent change of R d/R 0, where R d is resistance after MYOC infusion and R 0 is baseline resistance before MYOC infusion. Significance was tested with a paired, two tailed t-test. Washout time in the perfusion culture system was calculated using t 1/2 = (v/i)ln(2), where t is time (minutes), v is volume (1100 μl in cultured anterior segments), and i is flow rate (2.5 μl/min). 
Immunohistochemistry
Tissue samples were fixed in 4% paraformaldehyde, dehydrated in a graded series of ethanol (75%, 85%, 95%, 100%), and embedded in paraffin. Sections 5 μm thick were mounted on Superfrost/Plus glass slides (Fisher, Pittsburgh, PA), baked at 60°C for 2 hours, and deparaffinized. Tissue was incubated at 95°C for 30 minutes (1 mM EDTA, pH 8.0), blocked (3% BSA, 0.01% Triton in PBS) and incubated with anti-MYOC antibody. FITC-conjugated anti-rabbit Ig was used as secondary antibody (Sigma, St. Louis, MO). Tissue was also stained with DAPI to highlight the nuclei (1 μg/ml). Tissue sections were viewed on a fluorescence microscope. 
Results
Purification of Recombinant MYOC
The N-terminal tag of six histidine amino acids allowed Ni-NTA affinity chromatography purification of pRSET-MYOC, pRSET-β-galactosidase and pRSET-B lysates (Fig. 1) . Although no unique 55-kDa band can be detected in the pRSET-MYOC lysate when compared with the pRSET-B control (Fig. 1A , lanes 1 and 3), a 55-kDa band can be seen after several washes and elution from the Ni-NTA agarose (Fig. 1A , lane 6). Several other bands are also present in this lane that were also present in the control pRSET-B lysate (Fig. 1A , lane 4). Densitometric analysis of the Ni-NTA affinity chromatography purified pRSET-MYOC proteins revealed that the 55-kDa protein was 25% to 30% of the total elution, depending on variable overproduction and solubility of the recombinant protein in independent purifications. This is greater than a 500-fold purification of the 55-kDa protein from its initial lysate. 
Western blot analysis confirmed that MYOC was present in the starting lysate and had been partially purified by Ni-NTA affinity chromatography (Fig. 1B , lane 3 and 6). This was supported by subsequent analysis using the anti-Xpress antibody that is directed against an epitope in the N-terminal tag of the recombinant MYOC protein (Fig. 1C , lane 3 and 6) and the bacterial expression controlβ -galactosidase (Fig. 1C ; lanes 3 and 5). Peptide sequence analysis of two peptides isolated from the 55-kDa protein verified it as MYOC (data not shown). 
Outflow Resistance
Twelve pairs of human anterior segments were infused with recombinant MYOC. The baseline outflow facility for eyes treated with MYOC was 0.13 ± 0.01 (mean ± SEM), whereas the fellow eyes infused with various control proteins was 0.14 ± 0.02 (mean ± SEM). Addition of recombinant MYOC resulted in a steady increase in IOP starting 2 hours after anterior chamber exchange (Fig. 2A ). This reached maximal within 12 hours and remained above baseline levels for 48 to 72 hours. At maximal IOP, anterior segments infused with recombinant MYOC had a mean increase in outflow resistance of 94%± 12% (n = 7), whereas that from the fellow eyes (infused with purified proteins from pRSET-B lysate) was 12% ± 9% (mean ± SEM; experimental versus control: P = 0.0005; n = 7; Fig. 2B ). 
To show specificity for MYOC activity, recombinant MYOC was compared with heat-denatured recombinant MYOC (25-μg samples from the same purification batch). In three pairs of anterior segments, recombinant MYOC increased outflow resistance 101% ± 25%, whereas heat-denatured MYOC increased outflow resistance 12% ± 10% (mean ± SEM; experimental versus control: P = 0.03; Fig. 2B ). Additional specificity was shown in two pairs of anterior segments where MYOC (104% ± 45%; mean ± SEM) increased outflow resistance over the paired eye that was infused with bacterially expressed recombinant β-galactosidase (6% ± 1%; mean ± SEM). 
Controls
To determine whether the elevated IOP from MYOC was a simple bulk protein effect, several different controls were performed (Table 1) . None of the control proteins raised IOP including bovine serum albumin. Human serum (2500 μg) and fetal bovine serum (25,000μ g) also failed to raise IOP. Only the addition of 65,000 μg human serum (>2500-fold increase over the amount of recombinant MYOC) raised outflow resistance by 49% and 122% (two anterior segments tested at this dose). 
Immunohistochemistry
Meshwork and trabecular cells appeared normal in both MYOC and control anterior segments when examined by light microscopy (7 pairs). This was confirmed by electron microscopy in one MYOC-infused anterior segment. Using anti-MYOC antibody, intense staining of recombinant MYOC was found in small clumps scattered throughout the uveal, corneoscleral, and juxtacanalicular region (Fig. 3A ). These results were confirmed with the anti-Xpress monoclonal antibody (recognizes the N-terminal tag of the recombinant MYOC; data not shown). Additional staining of the sections with DAPI showed intact nuclei (Fig. 3B) . The control meshwork showed no staining for recombinant MYOC (Fig. 3C) . Control sections incubated with anti-rabbit Ig (secondary antibody but no primary antibody) showed no staining (Fig. 3D)
Discussion
Recombinant MYOC can be expressed and partially purified from a bacterial system. The addition of an N-terminal tag containing six histidine amino acids allowed purification using nickel ion affinity chromatography. This was essential since soluble expression levels of MYOC in the bacterial system were low, consisting of only approximately 0.01% of the total protein. 
The infusion of recombinant MYOC into anterior segments of human eyes resulted in an increase in IOP, starting 2 hours after addition and reaching maximal IOP within 12 hours. Several control proteins were run to determine whether this effect was specifically due to MYOC or could be a nonspecific effect of any protein. Bovine serum albumin, bacterially expressed β-galactosidase, fetal bovine serum, or human serum did not raise IOP even in amounts 10- to 100-fold higher than the recombinant MYOC dosage. Human serum in amounts up to 100-fold higher did not raise IOP, whereas amounts of >2500-fold over the amount of MYOC did increase IOP. The time course of the pressure elevation after human serum addition differed from that of MYOC. Maximal IOP was reached within 12 hours and returned to near baseline levels within 24 hours after human serum addition, whereas recombinant MYOC maintained elevated IOP for up to 72 hours after addition to anterior segments. The gradual elevation of IOP seen with MYOC (Fig. 2A) , occurring over 12 hours, indicates that MYOC slowly accumulated within the meshwork with the ongoing perfusion of media. At a flow rate of 2.5 μl/min and an anterior chamber washout time of t 1/2 = 5.1 hours, we calculated that approximately 20% of our recombinant MYOC preparation (5 μg) had entered the trabecular meshwork after 2 hours. In contrast, a much greater amount of human serum proteins were needed to see the same effect, and this lasted a shorter duration. This suggests that the interaction of recombinant MYOC within the meshwork is due to a specific property of MYOC and not a bulk protein effect. 
The infused MYOC was found mainly in the uveal region and anterior region of the trabecular meshwork. Lesser amounts were seen in the corneoscleral and juxtacanalicular regions (Fig. 3) . This distribution is consistent with the filtration function of the meshwork in removing debris from the anterior chamber. It does not necessarily indicate that these regions are involved in outflow resistance. Given that our calculations indicate as little as 5 μg of MYOC could elevate IOP, immuno-gold electron microscopy will be required to examine the juxtacanalicular and Schlemm’s canal region. 
A bacterial expression system was chosen for this initial study because of the rapid bacterial growth and relatively high amounts of recombinant protein that can be produced. The expression of eukaryotic proteins in a bacterial system, however, does not modify proteins with phosphate or sugars because bacteria do not have the capacity to perform these processes. Because the present study used a 55-kDa unmodified form of MYOC, the effect of such posttranslational modifications on MYOC cannot be determined. Furthermore, it is possible that protein folding differences exist between bacterial expressed MYOC and native MYOC. The native structure of MYOC is unknown, and hence it is unknown if our bacterially derived MYOC is folded similarly to the native protein. The finding that heat-denatured MYOC, which presumably is unfolded, did not increase pressure, whereas non–heat-treated MYOC from the same purification batch did increase pressure is an indication that the bacterial expressed MYOC was indeed folded. This is supported by our preliminary results of increased pressure when using MYOC secreted from a eukaryotic cell expression system (n = 2; data not shown). This eukaryotic expression system is expected to fold and process the protein normally. Until the conformation and function of native MYOC is known, the accuracy of any expression system will be open to interpretation. 
Could normal MYOC (nonmutant) cause elevated pressure in the human eye? The presence of MYOC in aqueous humor suggests that MYOC is found extracellularly and passes through the outflow pathway. 15 In addition, glucocorticoids induce expression of secreted MYOC in trabecular cells in both monolayer and perfusion organ culture in a time-dependent manner, similar to the time course for the development of steroid glaucoma. 16 Excessive amounts of extracellular MYOC, as may occur after steroid treatment, could potentially elevate IOP. The findings of the present study support this hypothesis. 
 
Figure 1.
 
Purification of recombinant MYOC. Starting lysates and purified samples were separated on a 10% SDS-PAGE gel. (A) GelCode Blue stained gel. (B) A duplicate gel containing the same samples as (A) were separated on a 10% SDS-PAGE gel and transferred to PVDF membrane. Western blot was probed with anti-MYOC antibody. (C) Western blot probed with anti-Xpress antibody. Lane 1, lysate from pRSET-B containing bacteria (20 μg); lane 2, lysate from pRSET-β-galactosidase containing bacteria (20 μg); lane 3, lysate from pRSET-MYOC containing bacteria (20 μg); lane 4, 250 mM imidizole elution of purified control proteins from bacteria transformed with pRSET-B (2 μ g); lane 5, 250 mM imidizole elution of purified β-galactosidase (2 μg); lane 6, 250 mM imidizole elution of purified recombinant MYOC (2 μg). Protein molecular weight standards are listed in kDa (Life Technologies, Grand Island, NY).
Figure 1.
 
Purification of recombinant MYOC. Starting lysates and purified samples were separated on a 10% SDS-PAGE gel. (A) GelCode Blue stained gel. (B) A duplicate gel containing the same samples as (A) were separated on a 10% SDS-PAGE gel and transferred to PVDF membrane. Western blot was probed with anti-MYOC antibody. (C) Western blot probed with anti-Xpress antibody. Lane 1, lysate from pRSET-B containing bacteria (20 μg); lane 2, lysate from pRSET-β-galactosidase containing bacteria (20 μg); lane 3, lysate from pRSET-MYOC containing bacteria (20 μg); lane 4, 250 mM imidizole elution of purified control proteins from bacteria transformed with pRSET-B (2 μ g); lane 5, 250 mM imidizole elution of purified β-galactosidase (2 μg); lane 6, 250 mM imidizole elution of purified recombinant MYOC (2 μg). Protein molecular weight standards are listed in kDa (Life Technologies, Grand Island, NY).
Figure 2.
 
Recombinant MYOC increases outflow resistance. (A) Graph indicating increase in IOP after addition of 25 μg of partially purified recombinant MYOC (black arrow, open squares). Partially purified control proteins (25 μg) isolated from bacteria containing pRSET-B expression vector were added to fellow eye (filled diamonds). (B) Effect on outflow resistance after addition of 25 μg of partially purified recombinant MYOC (n = 12), partially purified control (n = 7), heat-denatured recombinant MYOC (n = 3), and β-galactosidase (n = 2) in donor eyes. Data were taken from maximal IOP for eye infused with recombinant MYOC. Graph in (A) is represented in (B) by eye set 7.
Figure 2.
 
Recombinant MYOC increases outflow resistance. (A) Graph indicating increase in IOP after addition of 25 μg of partially purified recombinant MYOC (black arrow, open squares). Partially purified control proteins (25 μg) isolated from bacteria containing pRSET-B expression vector were added to fellow eye (filled diamonds). (B) Effect on outflow resistance after addition of 25 μg of partially purified recombinant MYOC (n = 12), partially purified control (n = 7), heat-denatured recombinant MYOC (n = 3), and β-galactosidase (n = 2) in donor eyes. Data were taken from maximal IOP for eye infused with recombinant MYOC. Graph in (A) is represented in (B) by eye set 7.
Table 1.
 
Perfusion of Human Anterior Segments
Table 1.
 
Perfusion of Human Anterior Segments
Sample Maximum Protein Dose (μg) Fold Increase IOP No. of Eyes
Recombinant MYOC 25 1 12
Purified control 25 1 NC 7
Recombinant MYOC (heat-denatured) 25 1 NC 3
Recombinant β-galactosidase 250 10 NC 2
Bovine serum albumin 250 10 NC 4
Fetal bovine serum 25,000 1000 NC 2
Human serum 65,000 >2500 2
Figure 3.
 
FITC immunohistochemistry of anterior segments infused with recombinant MYOC. Paraffin-embedded sections of anterior segments of the eye fixed 17 hours after addition of recombinant MYOC. (A) MYOC-infused; incubated with anti-MYOC. Bright green fluorescent speckles in uveal, corneoscleral, and JCT regions are recombinant MYOC. (B) DAPI stain of same section in (A). (C) Control eye infused with pRSET-B purified control proteins and incubated with anti-MYOC antibody. (D) MYOC-infused eye; no primary antibody, but incubation with FITC-conjugated anti-rabbit Ig secondary antibody. SC, Schlemm’s canal; AC, anterior chamber. Magnification, ×400.
Figure 3.
 
FITC immunohistochemistry of anterior segments infused with recombinant MYOC. Paraffin-embedded sections of anterior segments of the eye fixed 17 hours after addition of recombinant MYOC. (A) MYOC-infused; incubated with anti-MYOC. Bright green fluorescent speckles in uveal, corneoscleral, and JCT regions are recombinant MYOC. (B) DAPI stain of same section in (A). (C) Control eye infused with pRSET-B purified control proteins and incubated with anti-MYOC antibody. (D) MYOC-infused eye; no primary antibody, but incubation with FITC-conjugated anti-rabbit Ig secondary antibody. SC, Schlemm’s canal; AC, anterior chamber. Magnification, ×400.
The authors gratefully acknowledge the technical support from Chris Charlesworth, PhD (Mayo Protein Core Facility) and Steve Zeismer (Mayo Immunohistochemistry Core). 
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Figure 1.
 
Purification of recombinant MYOC. Starting lysates and purified samples were separated on a 10% SDS-PAGE gel. (A) GelCode Blue stained gel. (B) A duplicate gel containing the same samples as (A) were separated on a 10% SDS-PAGE gel and transferred to PVDF membrane. Western blot was probed with anti-MYOC antibody. (C) Western blot probed with anti-Xpress antibody. Lane 1, lysate from pRSET-B containing bacteria (20 μg); lane 2, lysate from pRSET-β-galactosidase containing bacteria (20 μg); lane 3, lysate from pRSET-MYOC containing bacteria (20 μg); lane 4, 250 mM imidizole elution of purified control proteins from bacteria transformed with pRSET-B (2 μ g); lane 5, 250 mM imidizole elution of purified β-galactosidase (2 μg); lane 6, 250 mM imidizole elution of purified recombinant MYOC (2 μg). Protein molecular weight standards are listed in kDa (Life Technologies, Grand Island, NY).
Figure 1.
 
Purification of recombinant MYOC. Starting lysates and purified samples were separated on a 10% SDS-PAGE gel. (A) GelCode Blue stained gel. (B) A duplicate gel containing the same samples as (A) were separated on a 10% SDS-PAGE gel and transferred to PVDF membrane. Western blot was probed with anti-MYOC antibody. (C) Western blot probed with anti-Xpress antibody. Lane 1, lysate from pRSET-B containing bacteria (20 μg); lane 2, lysate from pRSET-β-galactosidase containing bacteria (20 μg); lane 3, lysate from pRSET-MYOC containing bacteria (20 μg); lane 4, 250 mM imidizole elution of purified control proteins from bacteria transformed with pRSET-B (2 μ g); lane 5, 250 mM imidizole elution of purified β-galactosidase (2 μg); lane 6, 250 mM imidizole elution of purified recombinant MYOC (2 μg). Protein molecular weight standards are listed in kDa (Life Technologies, Grand Island, NY).
Figure 2.
 
Recombinant MYOC increases outflow resistance. (A) Graph indicating increase in IOP after addition of 25 μg of partially purified recombinant MYOC (black arrow, open squares). Partially purified control proteins (25 μg) isolated from bacteria containing pRSET-B expression vector were added to fellow eye (filled diamonds). (B) Effect on outflow resistance after addition of 25 μg of partially purified recombinant MYOC (n = 12), partially purified control (n = 7), heat-denatured recombinant MYOC (n = 3), and β-galactosidase (n = 2) in donor eyes. Data were taken from maximal IOP for eye infused with recombinant MYOC. Graph in (A) is represented in (B) by eye set 7.
Figure 2.
 
Recombinant MYOC increases outflow resistance. (A) Graph indicating increase in IOP after addition of 25 μg of partially purified recombinant MYOC (black arrow, open squares). Partially purified control proteins (25 μg) isolated from bacteria containing pRSET-B expression vector were added to fellow eye (filled diamonds). (B) Effect on outflow resistance after addition of 25 μg of partially purified recombinant MYOC (n = 12), partially purified control (n = 7), heat-denatured recombinant MYOC (n = 3), and β-galactosidase (n = 2) in donor eyes. Data were taken from maximal IOP for eye infused with recombinant MYOC. Graph in (A) is represented in (B) by eye set 7.
Figure 3.
 
FITC immunohistochemistry of anterior segments infused with recombinant MYOC. Paraffin-embedded sections of anterior segments of the eye fixed 17 hours after addition of recombinant MYOC. (A) MYOC-infused; incubated with anti-MYOC. Bright green fluorescent speckles in uveal, corneoscleral, and JCT regions are recombinant MYOC. (B) DAPI stain of same section in (A). (C) Control eye infused with pRSET-B purified control proteins and incubated with anti-MYOC antibody. (D) MYOC-infused eye; no primary antibody, but incubation with FITC-conjugated anti-rabbit Ig secondary antibody. SC, Schlemm’s canal; AC, anterior chamber. Magnification, ×400.
Figure 3.
 
FITC immunohistochemistry of anterior segments infused with recombinant MYOC. Paraffin-embedded sections of anterior segments of the eye fixed 17 hours after addition of recombinant MYOC. (A) MYOC-infused; incubated with anti-MYOC. Bright green fluorescent speckles in uveal, corneoscleral, and JCT regions are recombinant MYOC. (B) DAPI stain of same section in (A). (C) Control eye infused with pRSET-B purified control proteins and incubated with anti-MYOC antibody. (D) MYOC-infused eye; no primary antibody, but incubation with FITC-conjugated anti-rabbit Ig secondary antibody. SC, Schlemm’s canal; AC, anterior chamber. Magnification, ×400.
Table 1.
 
Perfusion of Human Anterior Segments
Table 1.
 
Perfusion of Human Anterior Segments
Sample Maximum Protein Dose (μg) Fold Increase IOP No. of Eyes
Recombinant MYOC 25 1 12
Purified control 25 1 NC 7
Recombinant MYOC (heat-denatured) 25 1 NC 3
Recombinant β-galactosidase 250 10 NC 2
Bovine serum albumin 250 10 NC 4
Fetal bovine serum 25,000 1000 NC 2
Human serum 65,000 >2500 2
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