Effect of protein conformation on rate of deamidation: ribonuclease A

The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68.     

Three-dimensional model and quaternary structure of the human eye lens protein gamma S-crystallin based on beta- and gamma-crystallin X-ray coordinates and ultracentrifugation.

A 3-dimensional model of the human eye lens protein gamma S-crystallin has been constructed using comparative modeling approaches encoded in the program COMPOSER on the basis of the 3-dimensional structure of gamma-crystallin and beta-crystallin. The model is biased toward the monomeric gamma B-crystallin, which is more similar in sequence. Bovine gamma S-crystallin was shown to be monomeric by analytical ultracentrifugation without any tendency to form assemblies up to concentrations in the millimolar range. The connecting peptide between domains was therefore built assuming an intramolecular association as in the monomeric gamma-crystallins. Because the linker has 1 extra residue compared with gamma B and beta B2, the conformation of the connecting peptide was constructed by using a fragment from a protein database. gamma S-crystallin differs from gamma B-crystallin mainly in the interface region between domains. The charged residues are generally paired, although in a different way from both beta- and gamma-crystallins, and may contribute to the different roles of these proteins in the lens.     

Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding

Calbindin D(28k) (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca(2+) homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37 degrees C or 50 degrees C in the presence or absence of Ca(2+). One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h(-1) at 50 degrees C for Ca(2+)-free calbindin. Deamidation is slower for the Ca(2+)-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca(2+) ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with beta-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca(2+) binding, i.e., the Ca(2+)-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca(2+) ions with high affinity in a parallel manner and with positive cooperativity.     

Effect of deamidation on folding of ribonuclease A

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65-72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn --> isoAsp where the local structure of the loop 65-72 has been modified keeping intact the C65-C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.     

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

Age‐related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long‐lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface‐exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen‐deuterium exchange, and susceptibility to disulfide cross‐linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light‐scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide‐linked aggregates. The lens‐specific chaperone αA‐crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS‐crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.     

Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system

Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4 h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2 days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fc-fusion protein. These are important considerations when evaluating the criticality of product quality attributes.     

Intrachain disulfide bond in the core hinge region of human IgG4.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.     

Disulfide bond reduction in fibrinogen: calcium protection and effect on clottability

Fibrinogen contains 29 disulfide bonds. Limited reduction in buffers containing calcium led to cleavage of three of them: the two A alpha 442Cys-A alpha 472Cys intrapeptide disulfide bonds and the symmetrical A alpha 28Cys-A alpha 28Cys bond. The limited reduction did not affect clotting by thrombin. However, a prolongation of the thrombin clotting time occurred when the limited reduction took place in the absence of calcium. The bonds reduced under this condition included the three already mentioned and also the two gamma 326Cys-gamma 339Cys intrapeptide disulfide bonds located in the C-terminal ends of the gamma-chain. N-Terminal analysis of thrombin-treated samples showed that thrombin cleavage occurred at the normal A alpha 16-A alpha 17 site in fibrinogen that was partially reduced in the presence of calcium. By contrast, thrombin cleaved at the A alpha 19-A alpha 20 site in fibrinogen that was partially reduced in the absence of calcium, rendering the protein unclottable by removing the A alpha 17Gly-18Pro-19Arg peptide. The loss of thrombin clottability may have also come from gamma 326Cys-gamma 339Cys disulfide bond reduction since the structure supported by this bond may be important for the function of the C-terminal polymerization site. In samples of the partially reduced fibrinogen lacking the A alpha 17-19 residues, gel formation occurred through an oligomerization mechanism catalyzed by factor XIII.     

High resolution TOF MS coupled to CE for the analysis of isotopically resolved intact proteins

Intact protein analysis by mass spectrometry is of great interest for the characterisation of biotechnological products. Exact mass measurement in combination with isotopic resolution allows the detection of modifications leading to small mass changes like deamidation or reduction of disulfide bonds directly on the level of the intact protein. Here, a concept is presented based on time-of-flight mass spectrometry. A bench top TOF MS and a high resolution TOF MS were used to resolve the isotopes of intact recombinant human growth hormone and intact human erythropoietin, respectively. Thus, these 22 and around 30kDa large proteins can be characterised sensitively in great detail and along with capillary electrophoretic separation unambiguous identification of minor protein modifications like deamidation is possible.     

Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoprotein D (gD) is a structural component of the herpes simplex virus envelope which is essential for virus penetration. The function of this protein is highly dependent on its structure, and its structure is dependent on maintenance of three intact disulfide bonds. gD contains six cysteines in its ectodomain whose spacing is conserved among all its homologs in other alphaherpesviruses as well as Marek's disease virus. For other proteins, conservation of cysteine spacing correlates with conservation of disulfide bond structure. We have now solved the disulfide bond structure of gD-1 and gD-2 of herpes simplex virus types 1 and 2, respectively. Two approaches were used. First, we constructed 15 double-Cys mutants of gD-1, representing all possible disulfide pairs. In each case, codons for cysteines were changed to serine. We reasoned that if two cysteines normally form a disulfide bond, double mutations which eliminate one proper bond should be less harmful to gD structure than double mutations which eliminate two disulfide bonds. The mutated genes were cloned into a eucaryotic expression vector, and the proteins were expressed in transiently transfected cells. Three double mutations, Cys-1,5, Cys-2,6, and Cys-3,4 permitted gD-1 folding, processing, transport to the cell surface, and function in virus infection, whereas 12 other double mutations each produced a malfolded and nonfunctional protein. Thus, the three functional double-Cys mutants may represent the actual partners in disulfide bond linkages. The second approach was to define the actual disulfide bond structure of gD by biochemical means. Purified native gD-2 was cleaved by CNBr and proteases, and the peptides were separated by high-performance liquid chromatography. Disulfide-linked peptides were subjected to N-terminal amino acid sequencing. The results show that cysteine 1 (amino acid [aa] 66) is bonded to cysteine 5 (aa 189), cysteine 2 (aa 106) is bonded to cysteine 6 (aa 202), and cysteine 3 (aa 118) is bonded to cysteine 4 (aa 127). Thus, the biochemical analysis of gD-2 agrees with the genetic analysis of gD-1. A similar disulfide bond arrangement is postulated to exist in other gD homologs.     

Molecular basis for the polymerization of octopus lens S-crystallin

S-Crystallin from octopus lens has a tertiary structure similar to sigma-class glutathione transferase (GST). However, after isolation from the lenses, S-crystallin was found to aggregate more easily than sigma-GST. In vitro experiments showed that the lens S-crystallin can be polymerized and finally denatured at increasing concentration of urea or guanidinium chloride (GdmCl). In the intermediate concentrations of urea or GdmCl, the polymerized form of S-crystallin is aggregated, as manifested by the increase in light scattering and precipitation of the protein. There is a delay time for the initiation of polymerization. Both the delay time and rate of polymerization depend on the protein concentration. The native protein showed a maximum fluorescence emission spectrum at 341 nm. The GdmCl-denatured protein exhibited two fluorescence maxima at 310 nm and 358 nm, respectively, whereas the urea-denatured protein showed a fluorescence peak at 358 nm with a small peak at 310 nm. The fluorescence intensity was quenched. Monomers, dimers, trimers, and polymers of the native protein were observed by negative-stain electron microscopic analysis. The aggregated form, however, showed irregular structure. The aggregate was solubilized in high concentrations of urea or GdmCl. The redissolved denatured protein showed an identical fluorescence spectrum to the protein solution that was directly denatured with high concentrations of urea or GdmCl. The denatured protein was readily refolded to its native state by diluting with buffer solution. The fluorescence spectrum of the renatured protein solution was similar to that of the native form. The phase diagrams for the S-crystallin in urea and GdmCl were constructed. Both salt concentration and pH value of the solution affect the polymerization rate, suggesting the participation of ionic interactions in the polymerization. Comparison of the molecular models of the S-crystallin and sigma-GST suggests that an extra ion-pair between Asp-101 and Arg-14 in S-crystallin contributes to stabilizing the protomer. Furthermore, the molecular surface of S-crystallin has a protruding Lys-208 on one side and a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp-179, and Asp-180) on the other side. We propose a molecular model for the S-crystallin polymer in vivo, which involves side-by-side associations of Lys-208 from one protomer and the aspartate patch from another protomer that allows the formation of a polymeric structure spontaneously into a liquid crystal structure in the lens.     

1H, 13C, and 15N assignments of wild-type human γS-crystallin and its cataract-related variant γS-G18V

We present the backbone and sidechain NMR assignments and a structural analysis of the 178-residue wild-type γS-crystallin and the cataract-related point mutant, γS-G18V. γS-crystallin is a structural component of the eye lens, which maintains its solubility and stability over many years. NMR assignments and continued structural investigations of γS-crystallin and aggregation-prone variants will advance understanding of cataract formation.     

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.     

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

ABSTRACT: BACKGROUND: Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. RESULTS: We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. CONCLUSIONS: This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.     

Specific glutamine and asparagine residues of gamma-S crystallin are resistant to in vivo deamidation

It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as gamma-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from gamma-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation.     

Monoclonal antibody OKT-9 recognizes the receptor for transferrin on human acute lymphocytic leukemia cells

The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of a disulfide bonded homodimer of m.w. 200,000 intact and 95,000 reduced. A similar protein is precipitated by transferrin-agarose, but not by agarose alone. Peptide mapping by limited proteolysis shows that the proteins precipitated by OKT-9 antibodies and transferrin-agarose are homologous. It is concluded that OKT-9 antibodies recognize the transferrin receptor. Expression of receptors for transferrin may be useful as a marker for activated or dividing cells.     

Facile cloning and sequencing of S-crystallin genes from octopus lenses based on polymerase chain reaction.

S-crystallin is a major lens protein present in the octopus and squid of Cephalopods. To facilitate the cloning of the protein, cDNA was constructed from the poly(A)+RNA of octopus lenses, and amplification by polymerase chain reaction (PCR) was carried out with two primers designed according to the 5'- and 3'-coding regions of S-crystallin gene. Sequencing two of 15 positive clones obtained shows 37-44% similarity in nucleotide and 23-30% similarity in amino acid sequences as compared with mammalian glutathione S-transferases (GST), revealing that S-crystallins exist as a multigene family and probably derived from GST by gene duplication and subsequent mutational base replacements.     

Nonclottable fibrin obtained from partially reduced fibrinogen: characterization and tissue plasminogen activator stimulation.

Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A alpha 442Cys-A alpha 472Cys and 2 gamma 326Cys-gamma 339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A alpha- and gamma-chains and the symmetrical disulfide bonds at gamma 8Cys, gamma 9Cys, and A alpha 28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A alpha 19-A alpha 20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A alpha-chain at A alpha 19-A alpha 20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma 326Cys-gamma 339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B beta 15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.     

The disulfide content of calf gamma-crystallin

The disulfide content of calf gamma-crystallin polypeptides has been investigated. The gamma-crystallin fraction of the soluble lens proteins was separated into five distinct polypeptides and characterized by isoelectric focusing, amino acid composition, and N-terminal sequence analysis to 25 residues. It has been demonstrated that 7 cysteines are present in gamma II, 4 to 5 cysteines in gamma IIIa, gamma IIIb, and gamma IV, and 6 cysteines in gamma I (beta s). Reduction of the total gamma-crystallin fraction with DTT resulted in an increase of approximately 1 to 1.5 mol of free SH per mole of protein. This increase in sulfhydryls was demonstrated to be contributed primarily by gamma II, the major polypeptide representing 50% of the total gamma-crystallin, which showed an increase of approximately 2.5 mol of sulfhydryl per mole of protein upon reduction. Insignificant disulfide content was present in gamma III and gamma IV and only a slight amount of disulfide was found in gamma I (beta s). The observed increase in sulfhydryl content upon reduction was not due to the presence of mixed disulfides of 2-mercaptoethanol, glutathione, or cysteine. The data are consistent with approximately 1 mol of intramolecular disulfide per mole of protein being present in gamma II. X-ray crystallography of gamma II has shown that the spatial location of Cys18 and Cys22 in the tertiary structure permits disulfide bond formation. Sequence analysis of the four major polypeptides of gamma-crystallin, gamma II, gamma IIIa, gamma IIIb, and gamma IV indicates that only gamma II has both Cys18 and Cys22. Cys18 is present in gamma IIIa, gamma IIIb, and gamma IV but Cys22 is replaced by His22. It is probable that the lack of disulfide in gamma IIIa, gamma IIIb, and gamma IV is due to the absence of Cys22.     

Complement component C8gamma is expressed in human fetal and adult kidney independent of C8alpha

Human complement component C8gamma is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily. So far, it has been found exclusively in plasma, covalently linked to C8alpha by disulfide bridging. We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8gamma. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8gamma is not dependent on C8alpha expression, since we could not detect C8alpha expression in kidney. Thus its physiological function is not restricted to a specific action in association with complement components. As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8gamma, we have expressed the encoding cDNA in Escherichia coli. To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm.