Hydrophobic interaction chromatography of recombinant human growth hormone, Genotropin.

A recombinant human growth hormone preparation (rhGH) has been used to study the effects of temperature, pH and detergent (Brij 35) concentration on the selectivity, recovery and retention time, during hydrophobic interaction chromatography (HI-HPLC) on a TSK-phenyl-5PW column. The rhGH preparation used in the study contained two rhGH variants, e.g. LMWGH and Clip I. These variants are structurally very similar to rhGH and exhibited very similar chromatographic behaviour to rhGH, important in evaluation of selectivity. Structural studies revealed that LMWGH had lost the first N-terminal amino acid residue, phenylalanine. Clip I exhibited an increased molecular mass of 32.7 Da for the C-terminal tryptic fragment T18-T19. Temperature and pH were found to influence retention time, sharpness of peaks and selectivity. Furthermore, recoveries were improved from 50% to 99% for rhGH upon the introduction of 0.075% Brij 35, a non-ionic detergent, in the presence of 5% acetonitrile. The optimized HI-HPLC system was found to exhibit good recoveries and excellent selectivity. The system is suitable both as an in-process chromatographic purification step for rhGH, as well as an analytical test method for purity and potency.     

Massspectrometrical analysis of recombinant human growth hormone Norditropin reveals amino acid exchange at M14_V14 rhGH

Recombinant human growth hormone (rhGH) is used for the treatment of several disorders. Structural integrity of rhGH is of critical importance for its clinical use and modifications thereof may act as markers in situations such as rhGH doping, as illegal rhGH-abuse in sports is of increasing interest. In the current study we investigated homogeneity of Norditropin, a recombinant human growth hormone frequently used in medicine, expressed in E. coli, strain MC1061. The most recent proteomics technologies including 2-DE, MALDI-MS followed by MALDI-MS/MS and LC-MS followed by LC-MS/MS were used for the characterisation of rhGH. MALDI-TOF-TOF and electrospray LC-MS analysis revealed one major protein with an average molecular mass of 22 126.0 Da and some additional minor components. Electrospray LC-MS/MS of the enzymatically digested Norditropin sample showed deamidation of N(12)N(149) and N(159), oxidation of M(14), M(125) and M(170) and one amino acid exchange V(14) for M(14) present in <1% of Norditropin. While deamidation and oxidation may be due to technical reasons, the single amino acid exchange may reflect infidelity of translation rather than codon usage and copy editing by E. coli.     

Location of amino acid substitutions in human hemoglobin. Mass spectrometric rapid analysis of tryptic peptides

Point mutations alpha 58 His----Tyr (Hb M Boston), beta 6 Glu--lys (Hb C) and beta 26 Glu----Lys (Hb E) have been identified in abnormal hemoglobins by means of tryptic hydrolysis of their alpha- and beta-chains followed by mass-spectrometry coupled with direct extraction of ions from solution. The abnormal hemoglobin Hb M Boston alpha 58 (E7) His----Tyr has been for the first time found in the blood of a patient from the USSR. This express-method is generally applicable for the identification of point mutation in proteins. The amount of protein necessary for the analysis is 100-1000 pmole. The stability, proteolytic degradation of the identified abnormal Hb's and Hb Bart's were investigated. The molecular pathogenesis of the hemoglobinopathies are discussed from the point of view of the observed properties.     

Mass spectrometrical analysis of the processed metastasis-inducing <Emphasis Type="Italic">anterior gradient protein 2 homolog</Emphasis> reveals 100% sequence coverage

Anterior gradient protein 2 homolog is a metastasis-inducing protein in a rat model of rat breast cancer and prognostic for outcome in hormonally treated breast cancer patients. Carrying out protein profiling in several mammalian cells and tissues, we detected this protein (synonym: secreted cement gland protein XAG-2 homolog) that was originally described in toad skin, in human bronchial epithelia. Tissues obtained from biopsies were homogenised and extracted proteins were run on two-dimensional gel electrophoresis. Following in-gel digestion with proteases trypsin, AspN, LysC and chymotrypsin, mass spectrometrical analysis was carried out by MALDI-TOF/TOF. The use of MS following multi-enzyme digestion of the protein resulted into 100% sequence coverage. MS/MS analysis enabled sequencing of 87% of the protein structure. This percentage does not include the signal peptide that was not observed in our protein due to processing. No posttranslational modifications were detectable and no sequence conflicts were observed. Complete analysis, unambiguous identification and characterisation of this biologically important protein could be shown, which is relevant for the definition of a marker protein that has been described so far by immunochemical methods only. Complete analysis is of importance as it forms the basis for all future work on this protein and, moreover, may serve as an analytical tool for further studies.     

Two types of amino acid substitutions in protein evolution

Summary The frequency of amino acid substitutions, relative to the frequency expected by chance, decreases linearly with the increase in physico-chemical differences between amino acid pairs involved in a substitution. This correlation does not apply to abnormal human hemoglobins. Since abnormal hemoglobins mostly reflect the process of mutation rather than selection, the correlation manifest during protein evolution between substitution frequency and physico-chemical difference in amino acids can be attributed to natural selection. Outside of abnormal proteins, the correlation also does not apply to certain regions of proteins characterized by rapid rates of substitution. In these cases again, except for the largest physico-chemical differences between amino acid pairs, the substitution frequencies seem to be independent of the physico-chemical parameters. The limination of the substituents involving the largest physicochemical differences can once more be attributed to natural selection. For smaller physico-chemical differences, natural selection, if it is operating in the polypeptide regions, must be based on parameters other than those examined.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Expression of the amino terminal part of synthetic human growth hormone gene and somatomedin-like activity of expressed protein

We have constructed three different plasmids containing parts of the human growth hormone gene using chemically synthesized oligomers and cloned them for the purpose of expressing them in Escherichia coli. AB, B and BC gene segments corresponding to ABhGH (residue 1-138), BhGH (residue 44-138) and BChGH (residue 44-192) were placed under the control of a tryptophan promoter in the expression vector. Upon induction with 3-indolylacrylic acid, ABhGH accumulated in cells but the BhGH and BChGH segments were not detected appreciably. Northern blotting analysis showed that the amount of mRNA transcribed from the AB gene segment was about ten-fold higher than that from the B or BC gene segment. ABhGH was found to have insulin-like growth factor I (IGF-I) activity, which could be explained by the hydrophilicity curves of these proteins.     

Mass spectrometry analysis of recombinant human ZP3 expressed in glycosylation-deficient CHO cells

The zona pellucida is an extracellular matrix that mediates taxon-specific fertilization in which human sperm will not bind to mouse eggs. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, ZP3). The primary structure of each has been deduced from the cDNA nucleic acid sequence, and each has been analyzed by mass spectrometry. However, determination of the secondary structure and processing of the human zona proteins have been hampered by the paucity of biological material. To investigate if taxon-specific sperm-egg recognition was ascribable to structural differences in a zona protein required for matrix formation, recombinant human ZP3 was expressed in CHO-Lec3.2.8.1 cells and compared to mouse ZP3. With nearly complete coverage, LC-QTOF mass spectrometry was used to determine the cleavage of an N-terminal signal peptide (amino acids 1-22) and the release of secreted ZP3 from a C-terminal transmembrane domain (amino acids 379-424). The resultant N-terminal glutamine was cyclized to pyroglutamate (pyrGln(23)), and several C-terminal peptides were detected, including one ending at Asn(350). The disulfide bond linkages of eight cysteine residues in the conserved zona domain were ascertained (Cys(46)/Cys(140), Cys(78)/Cys(99), Cys(217)/Cys(282), Cys(239)/Cys(300)), but the precise linkage of two additional disulfide bonds was indeterminate due to clustering of the remaining four cysteine residues (Cys(319), Cys(321), Cys(322), Cys(327)). Three of the four potential N-linked oligosaccharide binding sites (Asn(125), Asn(147), Asn(272)) were occupied, and clusters of O-glycans were observed within two regions, amino acids 156-173 and 260-281. Taken together, these data indicate that human and mouse ZP3 proteins are quite similar, and alternative explanations of taxon-specific sperm binding warrant exploration.     

Conformational effects of selected cancer-related amino acid substitutions in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Cancer-related mutations in the human p53 protein tend to cluster in four of the five highly conserved domains of the protein, and, in particular, in the central region of domain IV from residues 241 to 253. Using conformational energy analysis based on ECEPP (Empirical Conformational Energies for Polypeptides Program), we have determined the preferred three dimensional structures for this tridecapeptide sequence for the human wild-type p53 protein and four cancer-related mutant p53 proteins (Ala 245, Ile 246, Trp 248, Ser 249). The results show that the mutant peptides adopt conformations that are distinctly different from that of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in murine wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of human mutant p53 proteins may be mediated by distinct local conformational changes in the protein.     

Expression system for synthesis and purification of recombinant human growth hormone in Pichia pastoris and structural analysis by MALDI-ToF Mass Spectrometry

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZalphaA vector under the control of AOX1 promoter, which included a polyhistidine-tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non-native N- and C-termini. Analyses of the affinity-purified rhGH product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a spectral peak at m/z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu-Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile-Glu-Gly-Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N-Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.     

The 20,000-dalton variant of human growth hormone: Location of the amino acid deletions

The 20,000-dalton variant of human growth hormone lacks a sequence of 15 amino acids normally found in the hormone. The deletion involves residues 32 through 46. The remainder of the molecule is identical to the larger form of the hormone. These results suggest that the variant is the product of a deletion mutation in the growth hormone gene.     

Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures.

Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures. The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH. Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution. Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity. Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate. Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A. Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone. Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed. The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL.     

Properties of recombinant Rhizomucor miehei lipase with amino acid substitutions of Phe94 in the substrate binding domain

The chain length specificity of Rhizomucor miehei lipase was altered by substituting Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. Three recombinant enzymes, Phe94Arg, Phe94Glu and Phe94Gln, were expressed in Pichia pastoris, purified and their ability to hydrolyse p-nitrophenyl esters and triacylglycerols of different chain length was studied.     

Identification of amino acid substitutions in the lipopeptide surfactin using 2D NMR spectroscopy

It is generally accepted that surfactin, being produced by various Bacillus subtilis strains, is a cyclic lipopeptide built from the heptapeptide L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu and a beta-hydroxy fatty acid with variable chain length of 13 - 15 carbon atoms. We investigated surfactin from Bacillus subtilis ATCC 21332 and OKB 105, dissolved in pyridine and methanol, with two-dimensional H NMR spectroscopy. In the NH-fingerprint region, 21 well resolved cross peaks are observed instead of the expected seven cross peaks for the given heptapeptide. We were able to assign all proton signals to 21 amino acids, to identify three heptapeptides, and thus to prove the existence of structural analogues of surfactin. In the major fraction A, the peptide sequence is as given above. In fractions B and C, the C-terminal leucine is replaced by valine and isoleucine, respectively.     

Reciprocal amino acid substitutions in the evolution of homologous peptides

Amino acid sequences of peptides are often inferred from their amino acid compositions by comparison with homologous peptides of known sequence. The probabilities are considered that by such an approach errors are made due to the occurrence of balanced double changes, i.e. reciprocal substitutions, between two homologous peptides of identical compositions. Formulae are derived for the calculation of these probabilities, depending on peptide length and evolutionary distance. However, such calculations requiring too much computer time, the probabilities for reciprocal substitutions are estimated by simulation of evolutionary changes in peptides. It can be concluded from the resulting data that for many purposes the possible errors in amino acid sequences partially inferred from amino acid compositions are acceptably small.     

Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein

Transmissible spongiform encephalopathies (TSEs) are caused by a unique infectious agent which appears to be identical with PrPSc, an oligomeric, misfolded isoform of the cellular prion protein, PrPC. All inherited forms of human TSEs, i.e., familial Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker syndrome, and fatal familial insomnia, segregate with specific point mutations or insertions in the gene coding for human PrP. Here we have tested the hypothesis that these mutations destabilize PrPC and thus facilitate its conversion into PrPSc. Eight of the disease-specific amino acid replacements are located in the C-terminal domain of PrPC, PrP(121-231), which constitutes the only part of PrPC with a defined tertiary structure. Introduction of all these replacements into PrP(121-231) yielded variants with the same spectroscopic characteristics as wild-type PrP(121-231) and similar to full-length PrP(23-231), which excludes the possibility that the exchanges a priori induce a PrPSc-like conformation. The thermodynamic stabilities of the variants do not correlate with specific disease phenotypes. Five of the amino acid replacements destabilize PrP(121-231), but the other variants have the same stability as the wild-type protein. These data suggest that destabilization of PrPC is neither a general mechanism underlying the formation of PrPSc nor the basis of disease phenotypes in inherited human TSEs.