MutS as a tool for mutation detection

MutS, a DNA mismatch-binding protein, seems to be a promising tool for mutation detection. We present three MutS based approaches to the detection of point mutations: DNA retardation, protection of mismatched DNA against exonuclease digestion, and chimeric MutS proteins. DNA retardation in polyacrylamide gels stained with SYBR-Gold allows mutation detection using 1-3 microg of Thermus thermophilus his6-MutS protein and 50-200 ng of a PCR product. The method enables the search for a broad range of mutations: from single up to several nucleotide, as mutations over three nucleotides could be detected in electrophoresis without MutS, due to the mobility shift caused by large insertion/deletion loops in heteroduplex DNA. The binding of DNA mismatches by MutS protects the complexed DNA against exonuclease digestion. The direct addition of the fluorescent dye, SYBR-Gold, allows mutation detection in a single-tube assay. The limited efficiency of T4 DNA polymerase as an exonuclease hampers the application of the method in practice. The assay required 300-400 ng of PCR products in the range of 200-700 bp and 1-3 microg of MutS. MutS binding to mismatched DNA immobilised on a solid phase can be observed thanks to the activity of a reporter domain linked to MutS. We obtained chimeric bifunctional proteins consisting of T. thermophilus MutS and reporter domains, like beta-galactosidase or GFP. Very low detection limits for beta-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.     

A bifunctional chimeric protein consisting of MutS and beta-galactosidase

A bifunctional protein consisting of MutS, a mismatch binding protein and a beta-galactosidase reporter domain has been constructed. The fusion of beta-galactosidase to the MutS C-terminus was obtained by cloning the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. Milligram amounts of this huge chimeric protein (217 kDa monomer) were purified from 1l of overexpressing E. coli cells using metal-chelate affinity chromatography. The mismatch binding properties of the fusion protein were confirmed by DNA mobility shift assay in polyacrylamide gels. Binding to biotinylated mismatched DNA immobilized on streptavidin microplates followed by colorimetric reaction with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), demonstrated both mismatch recognition and beta-galactosidase activity of the chimeric protein. The activity of beta-galactosidase domain of the fusion was similar to that of the native enzyme. A colorimetric assay for beta-galactosidase activity using X-Gal supplemented with NBT (nitro blue tetrazolium) allowed detection of 50 and 500 fmol of the chimeric protein with naked eye in 45 microl volumes after 120 and 15 min incubation, respectively.     

Targeting expression of the catalytic domain of the kinase insert domain receptor (KDR) in the peroxisomes of Pichia pastoris

The kinase insert domain receptor (KDR), also known as vascular endothelial growth factor receptor-2 (VEGFR2), is an important therapeutic target for the treatment of cancer because of its crucial role in angiogenesis, which is fundamental to the malignancy of tumors. Here, we expressed the catalytic domain of KDR in Pichia pastoris under the control of the AOX1 promoter. In order to facilitate its purification and detection, His-tag and green fluorescent protein (GFP) were fused to the N-terminus of KDR. At the same time, a peroxisomal targeting signal 1 (SKL) was fused to the C-terminus to avoid the potential negative effect on the host cell. The highly expressing clone K1 was selected by GFP fluorescence intensity analysis using flow cytometry (FCM). Furthermore, the GFP-KDR-SKL fusion protein was proved to be correctly targeted to the peroxisomes of P. pastoris by colocation with blue fluorescent protein-SKL. The expression of GFP-KDR-SKL led to extensive phosphorylation of endogenous proteins and significantly inhibited cell growth. However, the expression was not lethal to the cells. Both in vitro biological activity assay and inhibition rate assay demonstrated that the purified GFP-KDR-SKL fusion protein exhibited high kinase catalytic activity and could be used as a target for anticancer drug screening.     

Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans.

The PrnB protein is a highly specific proline transporter that belongs to an amino acid transporter family conserved in both prokaryotes and eukaryotes. In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions. Several prnB-gfp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant. Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, four, or eight amino acids, with low potential to form secondary structure elements, were shown to be functional in vivo. A two-linker fusion results in partial complementation at both 25 and 37 degrees C. A four-linker fusion affords almost full complementation at 25 degrees C but partial complementation at 37 degrees C, whereas the eight-linker fusion results in partial complementation at both temperatures but in no GFP fluorescence. These results show that the number of linker amino acids is critical for the correct expression and/or translocation of PrnB-GFP fused proteins to the plasma membrane and for the fluorescence of the GFP. The expression of the four-linker PrnB-GFP transporter was detected and analyzed in vivo by both conventional fluorescence and confocal laser microscopy. This chimeric protein is localized in the plasma membrane, secondarily in large vacuoles found in the swollen conidial end of the germlings, and in other small intracellular structures observed as fluorescent granules. A strong correlation between known patterns of PrnB expression and intensity of PrnB-GFP fluorescence was observed. This work also demonstrates that the GFP fusion technology is a unique tool with which to study the expression and cellular localization of low-abundance transmembrane transporters expressed from their native promoters.     

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

Partial recovery of cell membrane-bounded human interleukin-2 fusion protein from insect cell debris by using various detergent extractions

We previously found that the human interleukin-2 (hIL-2) fused with green fluorescent protein (GFP) mainly remained in the insect cell debris after disruption due to the highly hydrophobic property of hIL-2 itself. Even though the significant GFPuv/hIL-2 fusion proteins were associated with cell membrane fractions, these were still functionally active. Therefore, to increase the total product yield, we performed partial recovery of the cell membrane-bounded hIL-2 fusion protein from the insoluble cell debris using several non-ionic, zwitterionic, and anionic detergents.     

烟曲霉蛋白质分泌载体的构建及酸性磷酸酯酶的细胞定位

[目的]在酵母细胞中蛋白质的糖基磷酸肌醇化(GPI)修饰是将GPI定位于细胞膜或细胞壁的信号.目前已对酵母GPI蛋白的细胞定位信号有一定了解,但对丝状真菌GPI蛋白的定位则了解甚少.AfPhoA是丝状真菌烟曲霉(Aspergillus fumigatus)的酸性磷酸酯酶,是GPI修饰的蛋白.该蛋白首先分离自细胞膜,随后又发现该蛋白与细胞壁结合.分析其C-端序列也未发现已知的定位信号,因此目前还不能确定其细胞定位.[方法]我们以绿色荧光蛋白(GFP)作为报告分子,将AfPhoA的C-端序列与GFP的C-端融合后检测融合GFP的细胞定位.[结果]我们用烟曲霉几丁质酶AfChiB1的启动子和N-端信号肽构建了可在烟曲霉中分泌表达GFP的表达载体pchiGFP.在此基础上将AfPhoA的C-端与GFP融合,融合质粒与pCDA14共转化烟曲霉后筛选到一株转化子.该转化子可表达融合GFP,在诱导和非诱导条件下,融合GFP均主要分布在细胞膜上,随培养时间的延长,融合GFP在细胞壁上也有少量分布;在培养上清液中只能检出约30KD的GFP融合蛋白,而没有完整的GFP融合蛋白,推测为从GPI锚上水解释放的.[结论]我们的研究结果表明,AfPhoA蛋白GPI修饰的作用是使该蛋白定位于细胞膜.本研究不仅初步确定了AfPhoA蛋白GPI修饰的细胞膜定位功能,而且为烟曲霉基因与蛋白质功能的研究建立了一个有效表达系统.     

Domain organization and functional analysis of Thermus thermophilus MutS protein.

MutS protein binds to DNA and specifically recognizes mismatched or small looped out heteroduplex DNA. In order to elucidate its structure-function relationships, the domain structure of Thermus thermophilus MutS protein was studied by performing denaturation experiments and limited proteolysis. The former suggested that T. thermophilus MutS consists of at least three domains with estimated stabilities of 12.3, 22.9 and 30.7 kcal/mol and the latter revealed that it consists of four domains: A1 (N-terminus to residue 130), A2 (131-274), B (275-570) and C (571 to C-terminus). A gel retardation assay indicated that T.thermophilus MutS interacts non-specifically with double-stranded (ds), but not single-stranded DNA. Among the proteolytic fragments, the B domain bound to dsDNA. On the basis of these results we have proposed the domain organization of T. thermophilus MutS and putative roles of these domains.     

Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli

AIMS: The thermal stability of isolated and extracted recombinant green fluorescent protein (GFPuv) was evaluated by analysing the loss of fluorescence intensity. METHODS AND RESULTS: GFPuv was expressed by Escherichia coli, extracted by the three-phase partitioning method and purified by elution through an hydrophobic interaction column. The collected fractions were further diluted in Tris-HCl-EDTA (pH 8.0) and subjected to continuous heating at set temperatures (45-95 degrees C). From a standard curve relating fluorescence intensity to GFPuv concentration, the loss of fluorescence intensity was converted to denatured GFPuv concentration (microg ml-1). To determine the extent of the thermal stability of GFPuv, decimal reduction times (D-values), z-value and energy of activation (Ea) were calculated. CONCLUSIONS: For temperatures between 45 and 70 degrees C, extracted native GFPuv activity decreased from 11 to 75% relative to initial native protein concentration above 70 degrees C, the average decrease in GFPuv fluorescence was between 72 to 83%. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermal stability of GFPuv provides the basis for its potential utility as a fluorescent biological indicator to assess the efficacy of the treatment of liquids and materials exposed to steam.     

Facile monitoring of avian reovirus σB expression and purification processes by tagged green fluorescent protein

Avian reovirus capsid protein σB was genetically fused with a histidine (His₆) tag and a UV-optimized green fluorescent protein (GFPuv) and expressed in Sf-9 cells. The fluorescence of GFPuv allowed for easy identification of protein localization and revealed that the fusion protein was quite stable in the cell culture. The fluorescence intensity (FI) exhibited a linear relationship (r² = 0.93) with the recombinant protein yield and therefore allowed for on-line tracking of the expression profile, which revealed an extremely high maximum yield of 70 μg per 10⁶ cells. The recombinant protein was purified via immobilized metal affinity chromatography (IMAC) and a high purity (85%) was achieved in one step. During the purification, the fluorescence again enabled qualitative and quantitative monitoring of when and how much the desired product was eluted. The GFP-tagging strategy eliminated the need for cumbersome and time-consuming assays (e.g. Western blot or ELISA) for product analysis, thus GFP is an effective non-invasive on-line marker for the expression and purification of recombinant proteins in the baculovirus expression system.     

Green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.     

Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.     

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.     

Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.     

Enhanced uptake of a heterologous protein with an HIV-1 Tat protein transduction domains (PTD) at both termini

Poor membrane permeability of proteins is a major limitation of protein therapy. In a previous study, we showed that the minimal sequence required for efficient transduction of Tat-GFP is the basic domain from 49-57 of HIV-1 Tat called the protein transduction domain (PTD. Here we have generated HIV-1 Tat PTD GFP fusion proteins in which HIV-1 Tat PTD is fused with the N- and/or C-termini of GFP. The various GFP fusion proteins were purified from Escherichia coli and characterized for their ability to enter mammalian cells using Western blot analysis, confocal microscopy and flow cytometry. The GFP fusion protein with Tat PTD at its C-terminus was taken up as efficiently as the GFP fusion protein with Tat PTD at its N-terminus. However, the same protein with PTDs at its both termini was taken up even more efficiently. All the GFP fusion proteins were present in both the nucleus and cytosol of the transduced cells. Uptake was lower at 4 degrees C than at 37 degrees C. The availability of the expression vectors developed in this study may help to devise novel strategies in the rational development of protein-based drugs.     

绿色荧光蛋白与Wee1 Hu融合基因的构建及其真核表达

 构建Wee1Hu与增强型绿色荧光蛋白 (GFP)融合基因表达载体 ,并对其在真核细胞的表达及生物学效应进行研究 .应用基因工程技术构建重组载体 ,脂质体转染胰岛 β细胞株 ,流式细胞仪和免疫沉淀 Western印迹检测融合蛋白的表达 ,共聚焦显微镜分析融合蛋白在活细胞内的分布 ,3 D结构模建分析其结构特点 ,并用MTT法检测其生物学活性 .结果显示融合基因在瞬时或稳定转染的真核细胞中均获表达 ,融合蛋白主要分布在胞核区 ,融合蛋白中Wee1Hu的空间构象与天然Wee1Hu完全相同 ,表达融合蛋白的胰岛β细胞可避免其被细胞毒T淋巴细胞 (CTL)杀伤 .结果表明GFP Wee1Hu融合蛋白中 ,可发绿色荧光的分子标签GFP未能影响Wee1Hu的结构及其生物学活性 ;Wee1Hu可通过调控细胞周期而阻断CTL介导的细胞凋亡     

Production of fluorescent single-chain antibody fragments in Escherichia coli

We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.     

Green fluorescent protein as a reporter for macromolecular localization in bacterial cells

Green fluorescent protein (GFP) is a highly useful fluorescent tag for studying the localization, structure, and dynamics of macromolecules in living cells, and has quickly become a primary tool for analysis of DNA and protein localization in prokaryotes. Several properties of GFP make it an attractive and versatile reporter. It is fluorescent and soluble in a wide variety of species, can be monitored noninvasively by external illumination, and needs no external substrates. Localization of GFP fusion proteins can be analyzed in live bacteria, therefore eliminating potential fixation artifacts and enabling real-time monitoring of dynamics in situ. Such real-time studies have been facilitated by brighter, more soluble GFP variants. In addition, red-shifted GFPs that can be excited by blue light have lessened the problem of UV-induced toxicity and photobleaching. The self-contained domain structure of GFP reduces the chance of major perturbations to GFP fluorescence by fused proteins and, conversely, to the activities of the proteins to which it is fused. As a result, many proteins fused to GFP retain their activities. The stability of GFP also allows detection of its fluorescence in vitro during protein purification and in cells fixed for indirect immunofluorescence and other staining protocols. Finally, the different properties of GFP variants have given rise to several technological innovations in the study of cellular physiology that should prove useful for studies in live bacteria. These include fluorescence resonance energy transfer (FRET) for studying protein-protein interactions and specially engineered GFP constructs for direct determination of cellular ion fluxes.     

Folding of the striated muscle myosin motor domain

We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1(795)GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25 degrees C and above the predominant product is an aggregate of S1(795)GFP or a complex with other lysate proteins. At 0 degrees C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1(795)GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg(2+)ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1(795)GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.