Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1(A2G))2 and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1(A2G))2-HSA, was over expressed under the control of promoter AOX1 and Mat alpha signal peptide in Pichia pastoris. SDS-PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1(A2G))2-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1(A2G))2-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1(A2G))2-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine.     

碳源对重组大肠杆菌发酵生产GLP-1融合蛋白的影响

以一株表达人胰高血糖素样肽-1融合蛋白的重组大肠杆菌为研究对象,首先通过摇瓶实验对碳源种类进行了初步选择,发现葡萄糖和甘油对菌体生长以及GLP-1融合蛋白表达较为适宜。进一步在5 L反应器上对初始葡萄糖及甘油浓度进行了考察,发现高浓度碳源有利于菌体生长却抑制GLP-1融合蛋白表达,但能提高GLP-1融合蛋白的体积得率。在0.25%初始葡萄糖或甘油存在的条件下,在培养过程中流加葡萄糖或甘油维持其在发酵液中的浓度,比较了两者对菌体生长以及GLP-1融合蛋白表达的影响,结果发现,以甘油为碳源时,菌体生长以及GLP-1融合蛋白的表达量均高于以葡萄糖为碳源的结果,最终发酵液的菌浓(OD_(600))可达到25.4,较葡萄糖为碳源时19.1提高了33.0%,GLP-1融合蛋白表达水平和体积得率分别可达到22.4%和1.051 g/L,较葡萄糖为碳源的15.8%和0.504 g/L分别提高41.8%和108.5%。该结果对GLP-1融合蛋白表达菌株发酵条件的进一步优化提供了依据。     

Construction of a Functional-Complementary Dual Insulinotropic Peptide rolGG and Its Therapeutic Effect on Type 2 Diabetes

Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) have a similar but complementary role in regulating blood glucose level. This study was aimed to develop a functional-complementary dual insulinotropic peptide which releases both GLP-1 and GIP in vivo, and to investigate its therapeutic effect on type 2 diabetes in mice. We firstly constructed a vector pET-22b(+)-rolGG to express a recombinant oral long-acting GLP-1?CGIP fusion peptide (rolGG) in E. coli. The rolGG peptide was then purified and confirmed its capacity of releasing recombinant oral long-acting GLP-1 (rolGLP-1) and recombinant GIP (rGIP) upon trypsin digestion in vitro, which were designed to resist in vivo enzymatic degradation. The therapeutic effect of rolGG was assessed in comparison with rolGLP-1 alone by daily oral-gavage administration up to 10?days in streptozotocin-induced type 2 diabetic mice. Saline and rosiglitazone administrations were served as negative and positive controls, respectively. The results showed that rolGG treatment decreased plasma glucose level by 26.7 and 46.3?% (p?<?0.01), respectively, at 5 and 10?days after the initial oral-gavage. The rolGG treatment also led to a trend of body weight increase, drink level and food intake decrease (p?<?0.05). In comparison, the oral administration of rolGLP-1 alone exhibited similar effects to rolGG with regard to plasma glucose level, drink level and food intake. In conclusion, we expressed and purified a dual insulinotropic peptide rolGG. Oral-gavage administration of rolGG showed a therapeutic effect on reduction of plasma glucose and alleviation of emaciation, polydipsia, and polyphagia symptoms in streptozotocin-induced diabetic mice.     

Identifying glucagon-like peptide-1 mimetics using a novel functional reporter gene high-throughput screening assay

Glucagon-like peptide-1 (GLP-1) stimulates insulin and inhibits glucagon secretion and therefore could potentially be used to treat diabetes type II. However, its therapeutic use is limited by its short half-life in vivo, due mainly to enzymatic degradation by dipeptidyl peptidase IV (DPP-IV). Developing GLP-1 analogs with greater bioactivity is therefore an important step toward using them therapeutically. Accordingly, we aimed to identify GLP-1 mimetic peptides by creating a high-throughput screening (HTS) assay of a phage displayed (PhD) peptide library. This assay was functionally based using the GLP-1 receptor (GLP-1R) gene. Rat GLP-1R cDNA was transfected into CHO/enhanced green fluorescent protein (EGFP) cells by lipofection. The resulting stable, recombinant cell line functionally expressed the GLP-1R and a cAMP-responsive EGFP reporter gene, to monitor receptor activation, and was used to screen a PhD dodecapeptide library. After four rounds of selection, 10 positive clones were selected based on functional evaluation and sequenced. Three sequences were obtained, corresponding to three different domains of GLP-1 (Group 1: 22-34; Group 2: 18-29; and Group 3: 6-17). The Group 3 peptide had the highest bioactivity, was synthesized, and designated KS-12. Importantly, KS-12 activated GLP-1R in vitro and reduced blood glucose levels in a dose-dependent manner when administered to Chinese Kunming mice. Although KS-12 was not as effective as GLP-1, it was significantly resistant to DPP-IV both in vitro and in vivo. Thus, this study provides a novel way to screen DPP-IV resistant agonist peptides of GLP-1 from a PhD peptide library using the functional reporter gene HTS assay.     

人GLP-1R基因转染BHK细胞及重组细胞的应用

为了构建表达人胰高血糖素样肽-1受体(GLP-1R)基因的BHK细胞株,并利用该重组细胞对GLP-1等相关肽进行活性测定,首先通过酶切、连接方式将人GLP-1R基因克隆至真核表达载体pCDNA3.(1 )中,然后用脂质体转染法将重组质粒转染至BHK-21细胞,转染后的细胞经G418加压筛选、细胞有限稀释等方法获得克隆细胞株。经过该细胞株RT-PCR验证,结果证实目的基因已整合至BHK-21细胞基因组中,并获得成功转录和表达。活性检测实验表明该重组细胞株经过GLP-1的刺激后,其细胞中的cAMP含量得到明显提升。该细胞株的构建为GLP-1及相关肽的活性测定奠定了基础。     

Expression and purification of exendin-4, a GLP-1 receptor agonist, in Escherichia coli

Exendin-4 is a 39 amino acid peptide isolated from salivary secretions of Gila monster (Heloderma suspectum). It shows 53% sequence similarity to glucagon-like peptide-1 (GLP-1), which is evaluated for the regulation of plasma glucose in type 2 diabetes. Exendin-4 is a potent and long-acting agonist of GLP-1 receptor. In the present study, the exendin-4 gene obtained by PCR with an enterokinase site at N-terminus and a termination codon at C-terminus was expressed in Escherichia coli strain BL21 (DE3) harboring pET32a(+). The fusion protein was purified by chromatography on Ni-NTA-agarose column. Recombinant exendin-4 was obtained by enterokinase cleavage of the fusion protein and subsequent purification. The yield of recombinant exendin-4 was 3.15mg/10g bacteria. The obtained recombinant exendin-4 shows glucose-lowering action in vivo.     

One-step purification of a fusion protein of glucagon-like peptide-1 and human serum albumin expressed in pichia pastoris by an immunomagnetic separation technique

Glucagon-like peptide-1 (GLP-1) has great therapeutic potential to treat diabetes type 2, mainly due to its unique glucose-dependent stimulation of insulin secretion profiles, but its clinical application is limited by its short half-life in vivo, which resultes from degradation by dipeptidyl peptidase IV and/or renal clearance. Developing long-acting GLP-1 analogs is therefore an important step toward using them therapeutically. In this study, the GLP-1/human serum albumin (HSA) fusion protein gene was cloned into the secretor type expression vector pPIC9K and subsequently expressed in Pichia pastoris. The expression quantity reached 58.5 mg/l in small-scale incubation. After optimization and characterization, the GLP-1/HSA fusion protein was successfully purified from the supernatant of the broth using immunomagnetic cellulose microspheres. HPLC showed that the purified GLP-1/HSA had an overall purity of 93.9%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the fusion protein exhibited the expected molecular mass of 70 kDa. Furthermore, that analysis of in vivo activity indicated that GLP-1/HSA reduced the blood glucose level after intraperitoneal administration to Chinese Kunming mice in a dose-dependent manner, and the effects held significantly 4 h after administration. Overall, this study illustrates the development of a long-acting GLP-1/HSA fusion protein expressed in Pichia pastoris.     

The N-terminal alanine-extended GLP-1/IgG-Fc fusion protein confers resistance to DPP-IV and reduces serum glucose level in db/db mice

The aim of this study was to develop novel long-acting glucagon-like peptide 1 (GLP-1) analogs resistant to dipeptidyl peptidase-IV (DPP-IV). We constructed three fusion proteins comprising GLP-1 and the human immunoglobulin gamma heavy chain (IgG-Fc); wild-type GLP-1 and IgG-Fc (GLP-1/IgG-Fc) and two N-terminal-extended fusion proteins in which an additional Ala (A) or Gly (G) was located on the N-terminus of GLP-1 (A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc). The fusion proteins expressed in CHO-K1 cells were secreted into medium and purified by Protein A affinity chromatography. Here, we show that the Ala or Gly-extended GLP-1/IgG-Fc fusion protein is resistant to DPP-IV and has increased half-life in vivo. To our surprise, the A-GLP-1/IgG-Fc fusion protein was more effective than wildtype GLP-1/IgG-Fc fusion protein in reducing blood glucose levels in db/db mice. Our findings suggest that the A-GLP-1/IgG-Fc fusion protein could be a potential long-acting GLP-1 receptor agonist for the treatment of insulin-resistant type 2 diabetes.     

Purification and bioactivity of exendin-4, a peptide analogue of GLP-1, expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Exendin-4, a peptide analogue of glucagon-like peptide-1 (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 fusion protein, expressed by methanol induction in Pichia pastoris system, was purified to homogeneity by chromatography followed by enterokinase cleavage of the fusion protein and subsequent purification of the recombinant exendin-4. Purity of the recombinant exendin-4 was 95.6%. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.     

重组胰高血糖素样肽-1与人血清白蛋白融合蛋白的纯化及活性研究

利用构建的重组菌株Pichia pastoris GS115/GLP-1/HSA,在10L发酵罐中表达了胰高血糖素样肽-1与人血清白蛋白融合蛋白(GLP-1/HSA),表达量为63.6mg/L。发酵液经中空纤维柱浓缩、疏水层析、阴离子交换层析和凝胶过滤分离纯化,获得了较高纯度的GLP-1/HSA,经HPLC分析纯度达95.8%。进一步的体内活性分析结果表明,GLP-1/HSA不仅具有天然GLP-1的生物活性,而且在给药后4h仍能发挥显著性降血糖作用。以上结果表明,利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的GLP-1/HSA,为进一步研究和开发能够用于糖尿病临床治疗的长效GLP-1类似物奠定了基础。     

Enzymic properties of recombinant BACE2.

BACE2 (Memapsin 1) is a membrane-bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the beta-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other 'general' aspartic proteases, the specificity of PB2-T2 is more constrained, apparently preferring to cleave at the NH2-terminal side of paired basic residues. Unlike other 'typical' aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1.     

Glucagon-like peptide-2-stimulated protein synthesis through the PI 3-kinase-dependent Akt-mTOR signaling pathway

Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation. GLP-2 stimulates mucosal growth in vivo with an increased rate of protein synthesis. However, it was unclear whether GLP-2 can directly stimulate protein synthesis. The objective was to test critically whether GLP-2 receptor (GLP-2R) activation directly stimulates protein synthesis through a PI 3-kinase-dependent Akt-mTOR signaling pathway. HEK 293 cells (transfected with human GLP-2R cDNA) were treated with human GLP-2 with/without pretreatment of PI 3-kinase inhibitor (LY-294002) or mTOR inhibitor (rapamycin). Results show that 1) GLP-2 specifically bound to GLP-2R overexpressed in the HEK cells with K(a) = 0.22 nM and B(max) = 321 fmol/μg protein; 2) GLP-2-stimulated protein synthesis was dependent on the amount of GLP-2R cDNA and the dosage of GLP-2 and reached the plateau among 0.2-2 nM GLP-2; 3) GLP-2-stimulated protein synthesis was abolished by the PI 3-kinase inhibitor and mTOR inhibitor; and 4) GLP-2-mediated stimulation of phosphorylation on Akt and mTOR was dependent on the amount of GLP-2R cDNA transfected and the dosage of GLP-2. In addition, GLP-2-mediated action and signaling in regulation of protein synthesis were confirmed in mouse hippocampal neurons (expressing native GLP-2R). GLP-2 directly stimulated protein synthesis of primary cultured neurons in dosage-dependent, PI 3-kinase-dependent, and rapamycin-sensitive manners, which linked with activation of Akt-mTOR signaling pathway as well. We conclude that GLP-2R activation directly stimulates protein synthesis by activating the PI 3-kinase-dependent Akt-mTOR signaling pathway. GLP-2-stimulated protein synthesis may be physiologically relevant to maintaining neuronal long-term potentiation and providing secondary mediators (namely neuropeptides or growth factors).     

重组胰高血糖素样肽-1与人血清白蛋白融合蛋白的纯化及活性研究

利用构建的重组菌株Pichia pastoris GS115/GLP-1/HSA,在10L发酵罐中表达了胰高血糖素样肽-1与人血清白蛋白融合蛋白（GLP-1/HSA）,表达量为63.6mg/L。发酵液经中空纤维柱浓缩、疏水层析、阴离子交换层析和凝胶过滤分离纯化,获得了较高纯度的GLP-1/HSA,经HPLC分析纯度达95.8%。进一步的体内活性分析结果表明,GLP-1/HSA不仅具有天然GLP-1的生物活性,而且在给药后4 h仍能发挥显著性降血糖作用。以上结果表明,利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的GLP-1/HSA,为进一步研究和开发能够用于糖尿病临床治疗的长效GLP-1类似物奠定了基础。     

Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo

Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.     

GLP-1 analogs containing disulfide bond exhibited prolonged half-life in vivo than GLP-1

The multiple physiological characterizations of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the therapy of type 2 diabetes. However, the half-life of GLP-1 is short in vivo due to degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. This indicates that the stabilization of GLP-1 is critical for its utility in drug development. In this study, we developed a cluster of GLP-1 mutants containing an inter-disulfide bond that is predicted to increase the half-life of GLP-1 in vivo. Exendin-4 was also mutated with a disulfide bond similar to the GLP-1 analogs. In this study, the binding capacities of the mutants were determined, the stabilities of the mutants were investigated and the physiological functions of the mutants were compared with those of wild-type GLP-1 and exendin-4 in animals. The results indicated that the mutants remarkably raised the half-life in vivo; they also showed better glucose tolerance and higher HbA_1c reduction than GLP-1 and exendin-4 in rodents. These results suggest that GLP-1 and exendin-4 mutants containing disulfide bonds might be utilized as possible potent anti-diabetic drugs in the treatment of type 2 diabetes mellitus.     

Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.     

Enzymatic- and renal-dependent catabolism of the intestinotropic hormone glucagon-like peptide-2 in rats

The intestinotropic hormone glucagon-like peptide (GLP)-2-(1-33) is cleaved in vitro to GLP-2-(3-33) by dipeptidyl peptidase IV (DP IV). To determine the importance of DP IV versus renal clearance in the regulation of circulating GLP-2-(1-33) levels in vivo, GLP-2-(1-33) or the DP IV-resistant analog [Gly(2)]GLP-2 was injected in normal or DP IV-negative rats and assayed by HPLC and RIA. Normal rats showed a steady degradation of GLP-2-(1-33) to GLP-2-(3-33) over time, whereas little or no conversion was detected for GLP-2-(1-33) in DP IV-negative rats and for [Gly(2)]GLP-2 in normal rats. To determine the role of the kidney in clearance of GLP-2-(1-33) from the circulation, normal rats were bilaterally nephrectomized, and plasma immunoreactive GLP-2 levels were measured. The slope of the disappearance curves for both GLP-2-(1-33) and [Gly(2)]GLP-2 were significantly reduced in nephrectomized compared with non-nephrectomized rats (P < 0.01). In contrast to both GLP-2-(1-33) and [Gly(2)]GLP-2, GLP-2-(3-33) did not stimulate intestinal growth in a murine assay in vivo. Thus the intestinotropic actions of GLP-2-(1-33) are determined both by the actions of DP IV and by the kidney in vivo in the rat.     

Functional importance of GLP-1 receptor species and expression levels in cell lines

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands.     

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins.

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.