Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:204–211, 2015     

Isolation and characterization of norleucine-resistant baby hamster kidney cells

Variants resistant to low and high levels of the methionine analogue norleucine were isolated in baby hamster kidney cells and in two clonal sublines, B1 and TG2. Clones resistant to high levels of norleucine were observed only after chemical mutagenesis, whereas clones capable of growing in low concentrations of norleucine occurred with equal frequency spontaneously and after mutagenesis. The variants were characterized with respect to uptake of ¹⁴C-norleucine and ¹⁴C-methionine. Five clones were found to be deficient in ¹⁴C-norleucine uptake, and of these, four showed reduced ¹⁴C-methionine uptake. The variants were tested also for increased activity of N₅-methyl-tetrahydrofolate: homocysteine methyltransferase, the enzyme which catalyses the terminal reaction in methionine biosynthesis. In four clones, higher levels of the methyltransferase were present than in the wild-type cells, suggesting overproduction or stabilization of this enzyme.     

Peak triceps surae muscle activity is not specific to knee flexion angles during MVIC

There is limited research on peak activity of the separate triceps surae muscles in select knee flexion (KF) positions during a maximum voluntary isometric contraction (MVIC) used to normalize EMG signals. The aim of this study was to determine how frequent peak activity occurred during an MVIC for soleus (SOL), gastrocnemius medialis (GM), and gastrocnemius lateralis (GL) in select KF positions, and if these peaks were recorded in similar KF positions. Forty-eight healthy individuals performed unilateral plantar-flexion MVIC in standing with 0°KF and 45°KF, and in sitting with 90°KF. Surface EMG of SOL, GM, and GL were collected and processed in 250 ms epochs to determine peak root-mean-square amplitude. Peak activity was most frequently captured in standing and rarely in sitting, with no position selective to SOL, GM or GL activity. Peak GM and GL activity was more frequent in 0°KF than 45°KF, and more often in similar KF positions than not. Peak SOL activity was just as likely in 45°KF as 0°KF, and more in positions similar to GM, but not GL. The EMG amplitudes were at least 20% greater in positions that captured peak activity over those that did not. The overall findings support performing an MVIC in more than one KF position to normalize triceps surae EMG. It is emphasized that no KF position is selective to SOL, GM, or GL alone.     

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.     

Mutational analysis of Saccharomyces cerevisiae nuclear RNase P: randomization of universally conserved positions in the RNA subunit.

Three regions in the Saccharomyces cerevisiae RNase P RNA have been identified, at positions Sce 87-94, Sce 309-316, and Sce 339-349, that contain nucleotides that are invariant in identity and position among all the known RNase P RNAs. To study the importance of these conserved RPR1 RNA regions in enzyme function, three independent mutational libraries were created in which the positions of invariant nucleotides were randomized simultaneously. Screening in vivo was used to identify viable RPR1 variants when reconstituted into holoenzyme in cells. Despite the universal evolutionary conservation, most of these positions tolerate certain sequence changes without severely affecting function. Most changes, however, produced subtle defects in cell growth and RNase P function, supporting the importance of these conserved regions. Isolation of conditional growth mutants allowed the characterization of the effects of mutations on cell growth, RPR1 RNA maturation, and activity of the holoenzyme in vitro. Kinetic analysis showed that viable variants were usually more defective in catalytic rate (Kcat) than in substrate recognition (Km).     

Critical amino acids of p21 protein are located within beta-turns: further evaluation

It has been shown that malignant activation of ras proto-oncogenes was mediated by point mutations which resulted in the single amino acid conversions at positions 12, 13 or 61 of the ras gene products (p21 proteins). By analyzing randomly mutated ras genes, it has been demonstrated that amino acid substitutions at residues 12, 13, 59 and 63 activated p21. Furthermore, it has been shown that residues 16, 116 and 119 in p21 played critical roles in the guanine nucleotide binding and, consequently, the ability of the protein to induce changes characteristic of cellular transformation. By using the protein conformational prediction method of Chou and Fasman, the present work predicts that these critical amino acids, except glutamic acid at position 63, are located within beta-turns. The major "hot spots" for ras activation are codons 12 and 61. The author has predicted in an earlier paper that the single amino acid conversions at positions 12 and 61 would occur at beta-turn conformation consisting of residues 10-13 and 58-61, respectively. In the present study, probabilities of beta-turn occurrence at residues 10-13 or 58-61 of the p21 proteins encoded by various ras genes are compared. The probability for the normal p21 containing glycine as residue 12 is greatest, and the cancer-associated variants show less probabilities. The single amino acid substitutions at position 61 do not cause so decreased probabilities of beta-turn potential at residues 58-61, except the replacement by histidine. Histidine at position 61 is not predicted as occurring within a beta-turn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methyl-Deficient Transfer Ribonucleic Acid and Macromolecular Synthesis in Methionine-Starved Saccharomyces cerevisiae

Haploid methionine auxotrophs of Saccharomyces cerevisiae continue to multiply for several hours after withdrawal of a required amino acid from the medium. Macro-molecular synthesis continues during this period of residual growth, although the net ribonucleic acid (RNA) and protein content is constant during the later part of this period. In this study, growth after withdrawal of methionine was in some cases accompanied by accumulation of transfer RNA (tRNA), which was shown by methylation in vitro to be deficient in methyl groups. This phenomenon was shown by only four of nine methionine auxotrophs tested, but no evidence could be found that these four strains had "relaxed" control of RNA synthesis. The nine methionine-requiring strains represent mutations in five different positions in the methionine biosynthesis pathway, and only mutants blocked at two of these five positions accumulated methyl-deficient tRNA. This accumulation therefore appears to be correlated with the position of the strain's block in the pathway of methionine biosynthesis.     

Methionine control of cephalosporin C formation

DL -Norleucine, a nonsulfur analogue of methionine was found to markedly stimulate synthesis of cephalosporin C by Cephalosporium acremonium strain CW19 in three different chemically defined media. Methionine, but not norleucine, stimulated cephalosporin C biosynthesis in a crude medium. The lack of stimulation by norleucine in complex medium was shown to be due to lack of uptake of this amino acid by mycelia growing in such a medium. In defined media containing a suboptimal methionine concentration, norleucine stimulated antibiotic production up to the level reached by optimal methionine. At an optimal dose of methionine, norleucine elicited no further increase in cephalosporin C production, indicating that these two amino acids act by the same mechanism. The data strongly indicate that stimulation by methionine is not a function of its ability to donate sulfur for antibiotic formation. Methionine was found to neither repress nor inhibit cysteine metabolism.     

Running-specific prostheses reduce lower-limb muscle activity compared to daily-use prostheses in people with unilateral transtibial amputations

People with unilateral transtibial amputation (TTA) have biomechanical differences between the amputated and intact legs and compared to people without TTA during running. Additional biomechanical differences emerge between running with running-specific (RSPs) and daily-use prostheses (DUPs), but the associated underlying muscle activity is unclear. We collected surface electromyography from the biceps femoris long head, rectus femoris, vastus lateralis, and gastrocnemius as well as body kinematics and ground reaction forces in six people with and six people without TTA. We compared stance phase muscle activity and peak activation timing in people with and without TTA and between people using RSPs compared to DUPs during running at 3.5 m/s. Peak amputated leg hamstring activity occurred 34% (RSP) and 31% (DUP) earlier in stance phase compared to the intact leg. Peak amputated leg rectus femoris activity of people wearing DUPs occurred 8% and 9% later in stance phase than the intact leg of people wearing DUPs and amputated leg of people wearing RSPs, respectively. People with TTA had 45% (DUP) and 61% (RSP) smaller peak amputated leg knee extension moments compared to people without TTA, consistent with observations of quadriceps muscle activity. Using RSPs decreased overall muscle activity compared to DUPs.     

NisinZ的定点突变及突变体性质的研究

以本实验室构建的含nisZ基因的质粒pHJ2 0 1为模板 ,采用定点突变技术将乳链菌肽Z分子中B环第 8位Thr突变为Ser(T8S)、将第 2位Dhb突变为Dha和第 31位His突变为Lys(T2S H31K)以及将第 2 7位Asn突变为Lys和第 31位His突变为Lys(N2 7K H31K) ,以pMG36e为载体 ,电击转化乳酸乳球菌 (L .lactis)NZ980 0进行表达。对表达产物性质的研究结果表明 ,3个突变体的抑菌谱和溶解度未发生变化 ,其抑菌活性略有下降 ,但它们的稳定性表现各不相同 :N2 7K H31K的稳定性与NisinZ几乎一致 ,而T8S和T2S H31K的稳定性有明显提高 ,在pH9条件下10 0℃加热 5min仍不丧失抑菌活性。     

Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering.

Nisin is a 3.4-kDa antimicrobial peptide that, as a result of posttranslational modifications, contains unsaturated amino acids and lanthionine residues. It is applied as a preservative in various food products. The solubility and stability of nisin and nisin mutants have been studied. It is demonstrated that nisin mutants can be produced with improved functional properties. The solubility of nisin A is highest at low pH values and gradually decreases by almost 2 orders of magnitude when the pH of the solution exceeds a value of 7. At low pH, nisin Z exhibits a decreased solubility relative to that of nisin A; at neutral and higher pH values, the solubilities of both variants are comparable. Two mutants of nisin Z, which contain lysyl residues at positions 27 and 31, respectively, instead of Asn-27 and His-31, were produced with the aim of reaching higher solubility at neutral pH. Both mutants were purified to homogeneity, and their structures were confirmed by one- and two-dimensional 1H nuclear magnetic resonance. Their antimicrobial activities were found to be similar to that of nisin Z, whereas their solubilities at pH 7 increased by factors of 4 and 7, respectively. The chemical stability of nisin A was studied in the pH range of 2 to 8 and at a 20, 37, and 75 degrees C. Optimal stability was observed at pH 3.0. Nisin Z showed a behavior similar to that of nisin A. A mutant containing dehydrobutyrine at position 5 instead of dehydroalanine had lower activity but was significantly more resistant to acid-catalyzed chemical degradation than wild-type nisin Z.     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

The fate of norleucine as a replacement for methionine in protein synthesis.

In vivo synthesised protein with norleucine occupying one half of the normal methionine loci was prepared using a methionine auxotroph of Escherichia coli K12. The extent of charging of the analogue onto both tRNA_met species and subsequent incorporation into soluble protein was monitored with a double-labelling system comprising [G-³H]norleucine and [³⁵S]methionine. Further experiments established that norleucine can be formylated in vivo once charged onto the initiator tRNA^f_met. An N-terminal analysis of the crude soluble protein revealed that formylnorleucyl-tRNA^f_met can initiate protein synthesis and that the formyl group is then removed from the nascent polypeptide. We were also led to conclude that the N-terminal methionine-amino peptidase does not recognise the analogue in this position. Slow growth rates on the methionine analogue have been partly attributed to limiting levels of charged tRNA^m_met, resulting in turn from the inefficiency of norleucine charging by methionyl-tRNA synthetase. Finally no evidence has been found for the production of aberrant protein as a result of norleucine incorporation, implying that limited growth on the analogue is due to its inability to replace methionine as the precursor of S-adenosyl methionine.     

In vitro construction of yeast tRNAAsp variants: nucleotide substitutions and additions in T-stem and T-loop.

A procedure for the construction of 3'-end labelled yeast tRNAAsp harboring substitutions or additions of any desired nucleotide in T-stem and T-loop (position 57 to 61) has been developed. This was done by in vitro enzymatic manipulations of the yeast tRNAAsp involving specific hydrolysis with RNases, phosphorylation and dephosphorylation with T4 polynucleotide kinase and ligation with T4 RNA ligase. Using this procedure we have replaced conserved or semi-conserved nucleotides located in position 57 to 61 of yeast tRNAAsp. We have also constructed different yeast tRNAAsp with eight bases instead of seven in T-loop. Further use of these tRNAAsp variants will be discussed with the help of the crystallographic three-dimensional structure.     

Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase

Escherichia coli grown in limited methionine and excess norleucine media accumulate cyanogen bromide-resistant species of proteins after the methionine supply is exhausted. Bacteria, transformed by recombinant plasmid pIPD37 carrying the adk gene and grown under limiting methionine and excess norleucine, synthesize 16-20% of adenylate kinase molecules having all 6 methionine residues replaced by norleucine. Species showing only partial replacement of methionine residues by norleucine are identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cyanogen bromide treatment of pure enzyme. Norleucine-substituted adenylate kinase shows structural and catalytic properties similar to the wild-type protein as indicated by circular dichroism spectroscopy and kinetic experiments but exhibits a much higher resistance to hydrogen peroxide inactivation under denaturing conditions.     

Comparison of two chemical cleavage methods for preparation of a truncated form of recombinant human insulin-like growth factor I from a secreted fusion protein

We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage.     

Relevance of cysteine residues for biosynthesis and antigenicity of human hepatitis B virus e protein.

The mature form of the secretory core protein (HBe protein) of human hepatitis B virus contains four cysteines which are located at amino acid positions -7, 48, 61, and 107 relative to the HBc start methionine. In addition, there is a cysteine, Cys-183, located in the C-terminal domain of the HBe precursor, which is cleaved during HBe maturation. Here, the significance of these cysteines for biosynthesis and antigenicity of the HBe protein was examined. The cysteines at positions -7 and 61 were found to be crucial for HBe biosynthesis. As has already been described, if the Cys at position -7 is mutated, disulfide-linked HBe homodimers which have both HBe antigenicity and HBc antigenicity are expressed. Here we show that these dimers are due to Cys-61-Cys-61 disulfide bridges which are formed only if the Cys at position -7 is not present. In the wild-type protein, this dimerization appears to be inhibited by formation of intramolecular disulfide bridges between the Cys at -7 and one of the internal cysteines. Moreover, Cys-61 is important for HBe biosynthesis in general since mutation of this amino acid results in production of HBe proteins which are either only poorly secreted or possess a different antigenicity.     

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.