Synthesis Of Recombinant Protein Research Articles

Construction and expression of Wee1 recombinant protein in Escherichia coli strain BL21 (DE3)

Wee1 is a gene encoding for protein kinase that is located in the nucleus and it plays an essential role in determining the timing of mitosis. Overexpression of Wee1 in rice is resulting in increased plant size. However, the increasing plant size due to the increase of Wee1 protein expression is not elucidated. Immunodiagnostic using specific antibodies against Wee1 protein should be conducted to determine the role of the protein on cell division. This experiment was directed to construct Wee1 in an expression vector for synthesis of recombinant Wee1 protein using three strategies of construction. The three strategies were construction of full length (FL), deletion of putative binding site (BS), and deletion of N-terminal domain (ΔN) of Wee1. Restriction analysis with BamH1/Sal1 of FL-Wee1, BS-Wee1, and ΔN-Wee1 construct resulted in the different DNA fragments with molecular size at 1239, 1176, and 960bp, respectively. This result indicated that the Wee1 fragments have been successfully inserted in the expression plasmid which was further confirmed using DNA sequencing. Colony PCR analysis showed that Escherichia coli strain BL21 (DE3) has been transformed with the constructs. The protein analysis using SDS-PAGE revealed that the recombinant protein of Wee1 synthesized in the E.coli containing ΔN-Wee1 construct, but not in FL-Wee1 and BS-Wee1 constructs. The ΔN-Wee1 protein was synthesized in an insoluble fraction with a molecular size of 38.8 kDa which is the same as the size estimated using the software ExPASy. Interestingly, the level of synthesized ΔN-Wee1 protein was not induced by IPTG concentration. Collectively, the results indicated that the DNA construct of ΔN-Wee1 is suitable for recombinant protein production.

Synthesis of Trimethyl Chitosan Nanoparticles Containing Recombinant BLF1-stxB Protein of Burkholderia Pseudomallei and Evaluation of its Immunogenicity in Mice

Synthesis of Trimethyl Chitosan Nanoparticles Containing Recombinant BLF1-stxB Protein of Burkholderia Pseudomallei and Evaluation of its Immunogenicity in Mice

An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein.

Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20°C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.

An efficient method for native protein purification in the selected range from prostate cancer tissue digests.

Prostate cancer (CP) cells differ from their normal counterpart in gene expression. Genes encoding secreted or extracellular proteins with increased expression in CP may serve as potential biomarkers. For their detection and quantification, assays based on monoclonal antibodies are best suited for development in the clinical setting. One approach to obtain antibodies is to use recombinant proteins as immunogen. However, the synthesis of recombinant protein for each identified candidate is time-consuming and expensive. It is also not practical to generate high quality antibodies to all identified candidates individually. Furthermore, non-native forms (e.g., recombinant) of proteins may not always lead to useful antibodies. Our approach was to purify a subset of proteins from CP tissue specimens for use as immunogen. In the present investigation, ten cancer specimens obtained from cases scored Gleason 3+3, 3+4 and 4+3 were digested by collagenase to single cells in serum-free tissue culture media. Cells were pelleted after collagenase digestion, and the cell-free supernatant from each specimen were pooled and used for isolation of proteins in the 10-30 kDa molecular weight range using a combination of sonication, dialysis and Amicon ultrafiltration. Western blotting and mass spectrometry (MS) proteomics were performed to identify the proteins in the selected size fraction. The presence of cancer-specific anterior gradient 2 (AGR2) and absence of prostate-specific antigen (PSA)/KLK3 were confirmed by Western blotting. Proteomics also detected AGR2 among many other proteins, some outside the selected molecular weight range, as well. Using this approach, the potentially harmful (to the mouse host) exogenously added collagenase was removed as well as other abundant prostatic proteins like ACPP/PAP and AZGP1 to preclude the generation of antibodies against these species. The paper presents an optimized scheme for convenient and rapid isolation of native proteins in any desired size range with minor modifications.

Synthesis and Characterization of Water-Soluble Silk Peptides and Recombinant Silk Protein Containing Polyalanine, the Integrin Binding Site, and Two Glutamic Acids at Each Terminal Site as a Possible Candidate for Use in Bone Repair Materials

The recombinant proteins [EE(A)12EETGRGDSPAAS]n (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea perni silk fibroin to which cells adhere well and combined poly(alanine), the integrin binding site TGRGDSPA, and a pair of glutamic acids (E2) at both the N- and C-terminal sites to render the construct water-soluble and with the hope that it might enhance mineralization with hydroxyapatite. Initially, two peptides E2(A)nE2TGRGDSPAE2(A)nE2 (n = 6, 12) were prepared by solid state synthesis to examine the effect of size on conformation and on cell binding. The larger peptide bound osteoblasts more readily and had a higher helix content than the smaller one. Titration of the side chain COO(-) to COOH of the E2 and D units in the peptide was monitored by solution NMR. On the basis of these results, we produced the related recombinant His tagged protein [EE(A)12EETGRGDSPAAS]n (n = 5,10) by expression in Escherichia coli . The solution NMR spectra of the recombinant protein indicated that the poly(alanine) regions are helical, and one E2 unit is helical and the other is a random coil. A molecular dynamics simulation of the protein supports these conclusions from NMR. We showed that the recombinant protein, especially, [EE(A)12EETGRGDSPAAS]10 has some of the properties required for bone tissue engineering scaffold including insolubility, and evidence of enhanced cell binding through focal adhesions, and enhanced osteogenic expression of osteoblast-like cells bound to it, and has potential for use as a bone repair material.

Class II ADP-ribosylation Factors Are Required for Efficient Secretion of Dengue Viruses

Identification and characterization of virus-host interactions are very important steps toward a better understanding of the molecular mechanisms responsible for disease progression and pathogenesis. To date, very few cellular factors involved in the life cycle of flaviviruses, which are important human pathogens, have been described. In this study, we demonstrate a crucial role for class II Arf proteins (Arf4 and Arf5) in the dengue flavivirus life cycle. We show that simultaneous depletion of Arf4 and Arf5 blocks recombinant subviral particle secretion for all four dengue serotypes. Immunostaining analysis suggests that class II Arf proteins are required at an early pre-Golgi step for dengue virus secretion. Using a horseradish peroxidase protein fused to a signal peptide, we show that class II Arfs act specifically on dengue virus secretion without altering the secretion of proteins through the constitutive secretory pathway. Co-immunoprecipitation data demonstrate that the dengue prM glycoprotein interacts with class II Arf proteins but not through its C-terminal VXPX motif. Finally, experiments performed with replication-competent dengue and yellow fever viruses demonstrate that the depletion of class II Arfs inhibits virus secretion, thus confirming their implication in the virus life cycle, although data obtained with West Nile virus pointed out the differences in virus-host interactions among flaviviruses. Our findings shed new light on a molecular mechanism used by dengue viruses during the late stages of the life cycle and demonstrate a novel function for class II Arf proteins.

A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene–T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C 3G 3 repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene–T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1 mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the “stress-responsive proteins”. Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.

Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein

The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within FRET-pair, the gene-engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21 (DE3) was studied and conditions of synthesis, isolation and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic light scattering. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of phosporescent spectroscopy. The obtained FRET-pair can be used both for studies in vitro and as a reporter in living cells.

Biological synthesis of alkyne-terminated telechelic recombinant protein

In this study, we demonstrate that the biological unnatural amino acid incorporation method can be utilizedin vivo to synthesize an alkyne-terminated telechelic protein. Synthesis of terminally-functionalized polymers such as telechelic polymers is recognized to be important, since they can be employed usefully in many areas of biology and material science, such as drug delivery, colloidal dispersion, surface modification, and formation of polymer network. The introduction of alkyne groups into polymeric material is particularly interesting since the alkyne group can be a linker to combine other materials using click chemistry. To synthesize the telechelic recombinant protein, we attempted to incorporate the L-homopropargylglycine into the recombinant GroES fragment by expressing the recombinant gene encoding Met at the codons for both N- and C-terminals of the protein in the Met auxotrophicE. coli via Hpg supplementation. The Hpg incorporation rate was investigated and the incorporation was confirmed by MALDI-TOF analysis of the telechelic recombinant protein.

Enhanced recombinant protein synthesis in batch and fed‐batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis

AbstractBACKGROUND: The formation of acetate as a metabolic by‐product in Escherichia coli fermentation is well known to have detrimental effects on cell growth and productivity. Various bioprocess and genetic approaches have previously been made to limit acetate accumulation, however, they tend to be conservative, limiting overall process productivity, or lead to other problems such as a decrease in maximum specific growth rate and decreased product yield on carbon.RESULTS: In this work, the utility of electrodialysis is examined as a potentially generic approach for in situ acetate removal and its impact on recombinant protein production. Using the induced synthesis of recombinant green fluorescent protein (GFP) in E. coli Tg1 (pGLO) as an example, it is shown that in situ removal of acetate to below inhibitory levels (∼1 g L−1) provides significant improvements in cell growth rate as well as specific biomass and recombinant protein yields. Experiments were performed in a 7.5 L stirred‐tank bioreactor using an external single cell‐pair electrodialysis module with an effective ion exchange membrane area of 0.01 m2. For this system increases in specific recombinant protein yield of up to 4‐fold have been observed dependent upon the time of induction, the mode of operation and the level to which acetate concentration is reduced in the fermentation broth.CONCLUSIONS: The implementation of ED can significantly increase the level of recombinant protein synthesis in batch and fed‐batch fermentation. The approach is considered to be generic, readily implemented and has wide application for the production of recombinant enzymes and proteins. Copyright © 2009 Society of Chemical Industry

Synthesis and evaluation of PDLs22 recombinant protein

Synthesis and evaluation of PDLs22 recombinant protein

Engineering eukaryotic signal transduction with RNAi: EnhancingDrosophila S2 cell growth and recombinant protein synthesis via silencing ofTSC1

RNAi has been useful in the study of biochemical pathways, but has not been widely used as a tool in metabolic engineering. The work described here makes use of double-stranded RNA (dsRNA) for the post-transcriptional gene silencing of TSC1 in Drosophila S2 cells. TSC1 downregulates the insulin-mediated signal transduction pathway, and serves as a metabolic control to guard against cellular overproliferation and tumorogenesis in both flies and mammals. By silencing TSC1 with in vitro-synthesized dsRNA, we have created a tunable and specific metabolic "throttle" that, like insulin, apparently increases the specific growth rate of S2 cells in a dose-dependent manner. This "throttle," augments the benefits of insulin addition while apparently avoiding deleterious and pleiotropic effects which can lead to lysis. During the period wherein dsRNA was active, cell growth rate was increased by 11% by the addition of 15 microg/mL dsTSC1 and by over 20% by the addition of 30 microg/mL dsTSC1. Additionally, synthesis of recombinant green fluorescent protein (GFP) was increased nearly 50% in a stable S2 cell line inducibly expressing GFP. Accordingly, we have "tuned" a normally tumorogenic pathway in animals into an advantage for both growth and recombinant product synthesis in cell culture. Potential applications for improving eukaryotic cell culture are anticipated.

Protein S secretion differences of missense mutants account for phenotypic heterogeneity

AbstractTo elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis. Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S. Modestly low protein S was found in families with R520G and P626L (group B) mutants. T57S and I518M (group C), which was associated with marginally low protein S, did not segregate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants. The 6 protein S mutants were transiently expressed in COS 1 cells. The Y444C mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%). Group B demonstrated around 50% reduction in secretion, whereas group C mutants showed normal secretion. Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants. Interestingly, there was a good correlation between in vitro secretion and the concentration of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency. (Blood. 2000;95:173-179)

Protein S secretion differences of missense mutants account for phenotypic heterogeneity

To elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis. Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S. Modestly low protein S was found in families with R520G and P626L (group B) mutants. T57S and I518M (group C), which was associated with marginally low protein S, did not segregate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants. The 6 protein S mutants were transiently expressed in COS 1 cells. The Y444C mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%). Group B demonstrated around 50% reduction in secretion, whereas group C mutants showed normal secretion. Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants. Interestingly, there was a good correlation between in vitro secretion and the concentration of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency. (Blood. 2000;95:173-179)

Enhanced production of human mini-proinsulin in fed-batch cultures at high cell density of Escherichia coli BL21(DE3)[pET-3aT2M2].

Synthesis of recombinant protein (human mini-proinsulin) is investigated in fed-batch cultures at high cell concentration of recombinant Escherichia coli BL21(DE3)[pET-3aT2M2]. Transcription of the recombinant gene is controlled by a T7 promoter system. The human mini-proinsulin is characterized by a C-chain peptide consisting of only nine amino acids, whereas the C-chain peptide of natural human proinsulin is made up of 35 amino acids. It is expressed in a fusion protein with a small fusion partner (a peptide with 18 amino acids) and finally aggregated into insoluble inclusion bodies in cytoplasm of recombinant E. coli. The fermentative production of this small fusion mini-proinsulin may be of great advantage in enhancing the yield of human insulin. To find an optimum induction strategy, effects of various key cultivation variables on the mini-proinsulin production are examined in high cell density fed-batch cultures. No general correlation is found between preinduction specific growth rate and recombinant protein synthesis, which confers a flexibility in choosing the feeding strategy of preinduction media for achieving the high cell density cultures. A culture temperature below 37 degrees C is unfavorable for recombinant gene expression, and the T7-based expression system is almost completely repressed at 30 degrees C. The nutrient glucose and yeast extract concentration in postinduction feed media is optimized by applying a statistical method for medium optimization, i.e. response surface methodology, and an effective amount of inducer molecule (IPTG) is determined to maximize the specific recombinant protein formation. The mini-proinsulin production in E. coli culture is significantly influenced by the volumetric feed rate of postinduction media, which is shown to be closely related to the plasmid copy number in the recombinant cell. Consequently, in a single-stage fed-batch process, the mini-proinsulin concentration is increased up to 7 g/L, approximately 62 wt % of which corresponds to mature human insulin. A two-stage fed-batch fermentation process, with recombinant cell growth occurring at a constant growth rate and constant cell concentration in a growth fermenter and mini-proinsulin production in an induction fermenter, is designed, and its efficacy in increasing volumetric productivity of mini-proinsulin is demonstrated.

Expression, Purification, and Characterization of Focal Adhesion Kinase Using a Baculovirus System

Focal adhesion kinase (FAK) has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus was generated which contains the mouse FAK cDNA cloned into a histidine tag transfer vector. Synthesis of the immunoreactive recombinant protein (baculovirus focal adhesion kinase (BFAK)Mr∼ 125,000) in infected Sf9 cells was detected 23 h postinfection, with maximal accumulation occurring at 48 h postinfection. BFAK constituted 5.4% of total soluble protein in the insect cell lysate and represented 19 mg/liter culture (∼2 × 109cells). The enzyme was active as a protein tyrosine kinase in both SF9 cells andin vitro.Purification to near homogeneity was achieved by nickel chelation chromatography. A yield of 5 mg of purified active BFAK was consistently produced from 1 liter of infected insect cells. BFAK tyrosine kinase activity was characterized physically using poly(Glu-Tyr) as a substrate. BFAK activity required the presence of a divalent cation and exhibited a preference for Mn2+over Mg2+. Maximal tyrosine kinase activity was attained at pH 7.2. Steady-state kinetic analysis with respect to ATP concentration did not conform to simple Michaelis–Menten kinetics and exhibited a Hill coefficient of much less than 1.Kmvalues for ATP using native and autophosphorylated BFAK were 6.7 ± 1.0 and 4.3 ± 0.2 μM, respectively.Kcatvalues were 13.9 ± 1.9 and 8.9 ± 0.3 nmol/min/mg BFAK. Steady-state kinetics with respect to the peptide substrate did fit the Michaelis–Menten equation and exhibited aKmvalue of 2.4 ± 0.3 μg/ml.

Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form

A quantitative study was performed to investigate the requirements for secretion of recombinant soluble and particulate forms of the envelope glycoprotein E of tick-borne encephalitis (TBE) virus. Full-length E and a carboxy terminally truncated anchor-free form were expressed in COS cells in the presence and absence of prM, the precursor of the viral membrane protein M. Formation of a heteromeric complex with prM was found to be necessary for efficient secretion of both forms of E, whereas only low levels of anchor-free E were secreted in the absence of prM. The prM-mediated transport function could also be provided by coexpression of prM and E from separate constructs, but a prM-to-E ratio of greater than 1:1 did not further enhance secretion. Full-length E formed stable intracellular heterodimers with prM and was secreted as a subviral particle, whereas anchor-free E was not associated with particles and formed a less stable complex with prM, suggesting that prM interacts with both the ectodomain and anchor region of E.

Control of growth and recombinant protein synthesis by heat-shock in a mutant mammalian cell line

The effect of heat-shock on cell growth and the induction of recombinant protein synthesis by a temperature-sensitive (ts) Chinese hamster ovary (CHO) cell line was investigated. An optimal regime of successive 2 hour heat-shocks (42°C) over 72 hours was found to simultaneously arrest cell growth and induce the synthesis of recombinant protein. The enhanced induction achieved from growth arrested cells may find application in the production of cytotoxic proteins.

Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coli

The introduction of random silent mutations into the 5−coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps. (1) Plasmid pUB200 containing the ϱ r ϱl promoters of phage λ was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA. (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein). (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids. When screening E. coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA. Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures. (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3. This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA. In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression.

Expression of variant von Willebrand factor (vWF) cDNA in heterologous cells: requirement of the pro-polypeptide in vWF multimer formation.

Von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized by vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula: (H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(OH) A heterologous expression system for the synthesis of recombinant vWF protein was developed, consisting of a monkey kidney cell line (COS-1), transfected with full-length vWF cDNA. This system was shown to mimic the constitutive secretory pathway of vWF in endothelial cells, since dimerization and multimerization occur similarly. To determine whether the pro-polypeptide, composed of the domains D1 and D2, is involved in vWF multimerization, a vWF cDNA was constructed that lacked the coding sequence for the pro-polypeptide. The mutant vWF protein, expressed by COS-1 cells transfected with this cDNA, did not assemble beyond the dimer stage. From this observation, we conclude that (i) dimerization does not involve the pro-polypeptide of pro-vWF and (ii) the presence of the pro-polypeptide, as part of pro-vWF, is obligatory for multimerization. It is argued that the interactions, required for interchain binding, are mediated by the D domains.