Activity and Biospecificity of Proteolyzed Forms and Dimeric Combinations of Recombinant Human and Murine Nerve Growth Factor

Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland-derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed-phase HPLC (RP-HPLC), and high-performance ion-exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120-residue form of rhNGF produced a species of 109 residues (10-118). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N-terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N-terminal sequence, PAGE, and RP-HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma-12 cell line neurite extension assay. The 118-residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal-modified molecules presented activity reduced by 50- to 100-fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.     

Physicochemical Characterization of Recombinant Human Nerve Growth Factor Produced in Insect Cells with a Baculovirus Vector

Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion-exchange and reversed-phase chromatography to near homogeneity. The N-terminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (less than 0.08 mol of N-acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF from insect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimer's disease.     

Primary structure of the southern bean mosaic virus coat protein: First results

Studies on the southern bean mosaic virus coat protein have established the molecular weight of this protein, its amino acid composition, the nature of its C-terminal amino acid, and the blockage of the N-terminal residue by an acetyl group. After hydrolysis of the protein by trypsin, the hydrolysate was fractionated by ion-exchange chromatography. Among the purified tryptic peptides were isolated the N- and the C-terminal peptides where sequences were determined, principally by mass spectrometry.     

Isolation and primary structure of trypsin from the red king crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the Paralithodes camtschaticus crab was isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. The cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the Penaeus vannamei shrimp appeared to be the most close in its primary structure to that of the king crab (70% identity).     

Isolation and amino acid sequence of two trypsin isoinhibitors from duck pancreas

DPTI II and DPTI IV, two trypsin inhibitors from duck pancreas, have been isolated by affinity chromatography on immobilized anhydrotrypsin, anion exchange and RP-HPLC. The complete amino acid sequence of both inhibitors was determined after reductive carboxymethylation and digestion with Staphylococcus aureus V8 protease or trypsin. The inhibitors were each found to be a single polypeptide chain comprised of 69 amino acid residues and their molecular masses were estimated at 7687 Da for DPTI II and 7668 Da for DPTI IV. The only difference in amino acid sequence between the two inhibitors is the replacement of Arg for His residue in the C-terminal position of DPTI IV.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.     

Luteinizing hormone of the sperm whale. Isolation, separation into subunits and study of the amino acid sequence of the alpha-subunit

The luteinizing hormone isolated from sperm-whale pituitary was separated into two subunits, alpha- and beta-, by ion-exchange chromatography on sulfoethyl-Sephadex. The hormone subunits were reconstituted, carboxymethylated and cleaved by BrCN and proteolytic enzymes. In order to block tryptic hydrolysis at lysine residues the alpha-subunit was subjected to maleylation. Large-sized fragments of BrCN were cleaved by chymotrypsin and trypsin, while large-sized fragments of trypsin were split by chymotrypsin. The resulting peptides were separated by gel filtration on Sephadex, ion-exchange chromatography on Aminex A-5 and thin-layer partition chromatography on cellulose. The amino acid sequence of the peptides was determined by the Edman method, using identification of the N-terminal amino acids in a reaction with dansyl chloride or dimethylaminoazobenzene-4-isothiocyanate. It was shown that the alpha-subunit of the luteinizing hormone is a peptide chain consisting of 96 amino acid residues with covalently linked carbon chains at asparagine residues at positions 56 and 82. The N-terminal amino acid of the alpha-subunit is phenylalanine, the C-terminal amino acid is serine. The alpha-subunit is heterogeneous at the N-end, i. e. beside phenylalanine it contains threonine and trace amounts of proline, aspartate, glutamate and glycine.     

Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.

Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.     

黑豆种子中一种耐热型胰蛋白酶抑制剂的分离及性质表征

应用硫酸铵分级沉降、弱阳交换色谱CM-Sephadex C-50、凝胶过滤 色谱Sephacryl S-200HR、强阳离子高效液相色谱POROS HS-20,从黑豆种 子中分离纯化一种耐热型蛋白酶抑制剂,命名为TSTI.该蛋白的Ｎ末端序列 为DEYSKPCCDLCMCTRRCPPQ,与豆科植物Bowman-Birk型胰蛋白酶抑制剂具有 高度同源性,推测其属于Bowman-Birk型胰蛋白酶抑制剂.SDS－PAGE和IEF 测出TSTI分子量和等电点分别为23.9 kD 和6.2.TSTI对胰蛋白酶有很强的 抑制作用,当二者摩尔比达到1时,胰蛋白酶活力被完全抑制.此外,该蛋 白酶抑制剂具有很强的热稳定性和pH稳定性,在高达100 ℃温度及pH2-12 范围内处理,其活性均不会受到太大影响.TSTI对植物致病菌苹果轮纹病菌 、瓜果腐霉病菌、白菜黑斑病菌、甜瓜枯萎病菌和葡萄灰霉病菌具有抑制 作用.     

A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.

A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors.     

Biochemical and structural comparative study between bird and mammal pancreatic colipases

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of approximately 10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Purification of limited tryptic fragments of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum and identification of conformation-sensitive cleavage sites

Sarcoplasmic reticulum membranes were treated with trypsin, and samples enriched with A1a, A1b, and C fragments (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), respectively, were prepared. A1b and C fragments were purified to apparent homogeneity, and an approximately equimolar mixture of A1(Met1-Arg198), A1a, and A1b fragments free from other contaminants was also obtained through gel permeation and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate. N- and C-terminal amino acid sequence analyses of these peptides were carried out in order to identify the tryptic cleavage sites responsible for the formation of these fragments. Both A1a and A1b fragments had the same C-terminal sequence as A1 fragment. Single cleavage of A1 at T3a (Lys218-Ala219) yielded A1a, while a cleavage between either Lys234-Ile235 or Arg236-Asp237 (collectively designated as T3b) resulted in A1b fragment. Thus, A1a and A1b fragments differed from A1 fragment only by their loss of short stretches corresponding to the N-terminal region of the latter. On the other hand, C fragment represented the C-terminal half of B fragment (Ala506-Gly994). It had the same C-terminal sequence as B fragment and was produced by cleavage at T4 (Lys728-Thr729). Cleavages at T3a and T3b profoundly affected the catalytic properties of SR-ATPase (Imamura, Y. and Kawakita, M. (1986) J. Biochem. 100, 133-141), and it was suggested that the segment of the ATPase molecule including the region between Ala199 and Arg236 is important in mediating the coupling between ATP splitting and Ca2+-transport.     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

猪胰蛋白酶自溶产物中δ-、γ-和σ-胰蛋白酶的分离与结晶

将1％猪胰蛋白酶溶于0.05M,pH9.0硼酸缓冲液中,在25℃自溶6小时后,上大豆胰蛋白酶抑制剂亲和层析柱STI-Sepharose4B,用pH5.0—3.0的磷酸钾缓冲液梯度洗脱,可以有效地分开各种自溶活性产物。从得到的三个活性峰S_1、S_2和S_3中,用DNS-Cl Edman法和有色Edman法测定酶分子肽键断裂后N末端的部分氨基酸顺序,从而鉴定出三种不同形式的自溶活性产物:δ~-、γ~-和σ~-胰蛋白酶。S_3主要含有完整的单链β-胰蛋白酶;S_2主要含有Arg_(105)—Val_(156)断裂的双链δ-胰蛋白酶;S_1含有Arg_(105)—Val_(106),Lys_(145)—Arg_(146)和Arg_(105)—Val_(106),Lys_(131)—Ser_(132)断裂的三链γ-和σ-胰蛋白酶。活性产物的几个N端氨基酸顺序与已知猪胰蛋白酶氨基酸顺序完全相符。 与β-胰蛋白酶相比,δ~-、γ~-和σ~-胰蛋白酶的等电点稍有降低,约为10.5左右。 对分离出的自溶活性产物的结晶条件进行了摸索,用悬滴法已经得到δ~-、γ~-和σ~-胰蛋白酶与苯甲脒复合物的结晶。这是关于分离出胰蛋白酶自溶活性产物结晶体的首次报道。     

Biochemical characterization and N-terminal sequences of two new trypsin inhibitors from Copaifera langsdorffii seeds

Two new trypsin inhibitors, TDI-I and TDI-II, were purified from the seeds of the native Brazilian tree Copaifera langsdorffii (Caesalpinoideae, Leguminosae). The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on trypsin-Sepharose, and reversed-phase (RP) HPLC. RP-HPLC yielded two forms (TDI-I and TDI-II), as confirmed by isoelectric focusing, with pI values between 7.0 and 8.1. The molecular mass of the TDI forms was 24 kDa based on FPLC gel filtration on Superdex 75. Under reducing conditions in tricine SDS-PAGE the molecular masses of TDI-I and TDI-II were 12 and 10 kDa, respectively. The Ki values were 1.1 and 1.2 nM for TDI-I and TDI-II, respectively, and there was no inhibitory effect on chymotrypsin. Amino acid analysis revealed high levels of aspartic acid, glutamic acid, serine, glycine, proline, and lysine but low levels of methionine and aromatic amino acids in both inhibitors; the calculated molecular masses were 11,456 and 10,008 for TDI-I and II, respectively. Based on the N-terminal sequences of TDI-I and TDI-II, TDI-I belongs to the Kunitz family of trypsin inhibitors, whereas TDI-II showed no homology to any other protein. This observation suggests that TDI-II belongs to a new inhibitor subclass of low-molecular mass proteins in the subfamily Caesalpinoideae.     

ISOLATION OF A TRYPSIN–CHYMOTRYPSIN INHIBITOR AND ITS FUNCTIONAL PROPERTIES

A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman–Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85°C, as well as within the pH range 2–12. Besides inhibiting trypsin–chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor's extraction and isolation for the first time, but also its physiochemical and antifungal properties.     

Role of C-terminal sequence of cytochrome P450scc in folding and functional activity

To elucidate the role of Arg472 and C-terminal sequence of the mature form of cytochrome P450scc, a mitochondrial cytochrome P450, in the present work we have performed sequential removal of the C-terminal amino acid residues of the hemeprotein and evaluated their functional role in folding and catalysis. The removal of 2, 4, 7, or 9 amino acid residues (cytochrome P450scc mutants Delta2, Delta4, Delta7, and Delta9) does not significantly affect the physicochemical properties of the truncated forms of cytochrome P450scc, but results in significant increase in the expression level of the hemeprotein in Escherichia coli (Delta4 cytochrome P450scc mutant). However, removal of 10 C-terminal amino acid residues (Delta10 cytochrome P450scc) of mature form of cytochrome P450scc (replacement of codon for Arg472 for stop-codon) is followed by loss of the ability for correct folding in E. coli. Based on these data, it is concluded that the C-terminal amino acid residues of cytochrome P450scc (DeltaArg472-Ala481) play an important role in correct recombinant protein folding and heme binding by cytochrome P450scc during its expression in E. coli, while folding of mitochondrial cytochrome P450scc during its heterologous expression in bacterial cells is more similar to the folding of prokaryotic soluble cytochrome P450's than to microsomal cytochrome P450's.     

Isolation and Primary Structure of Trypsin from the King Crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the crab Paralithodes camtschaticuswas isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. A cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the shrimp Penaeus vannamei appeared to be closest in primary structure to that of the king crab (65% identity).