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Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
The primary structure is held together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. These peptide bonds provide rigidity to the protein. The two ends of the amino acid chain are referred to as the C-terminal end or carboxyl terminus (C-terminus) and the N-terminal end or amino terminus (N-terminus) based on the nature of the free group on each extremity. The various types of secondary structures are defined by their patterns of hydrogen bonds between the main-chain peptide groups. However, these hydrogen bonds are generally not stable by themselves, since the water—amide hydrogen bond is generally more favorable than the amide—amide hydrogen bond. Thus, the secondary structure is stable only when the local concentration of water is sufficiently low, for example, in the molten globule or fully folded states. Similarly, the formation of molten globules and tertiary structure is driven mainly by structurally nonspecific interactions such as the rough propensities of the amino acids and hydrophobic interactions. However, the tertiary structure is fixed only when the parts of a protein domain are locked into place by structurally specific interactions, which include ionic interactions (salt bridges), hydrogen bonds, and the tight packing of side chains. The tertiary structure of extracellular proteins can also be stabilized by disulfide bonds, which reduce the entropy of the unfolded state. Disulfide bonds are extremely rare in cytosolic proteins since the cytosol is generally a reducing environment.
Enzyme Catalysis
Published in Harvey W. Blanch, Douglas S. Clark, Biochemical Engineering, 1997
Harvey W. Blanch, Douglas S. Clark
In some cases, accumlation of a eukaryotic protein expressed directly in E. coli is limited because the protein is recognized as foreign and is degraded. However, expression levels can sometimes be improved by linking a eukaryotic gene with a bacterial gene to produce a so-called fusion protein. If the end use of the final product requires that the eukaryotic polypeptide be isolated from the fusion protein, a cleavable peptide sequence, often called a linker peptide, can be inserted between the C-terminus of the prokaryotic sequence and the N-terminus of the eukaryotic sequence. Cleavage can then be effected by chemical methods or by proteolytic enzymes. Another fusion method is one in which multiple copies of the gene are linked in tandem. For example, enhanced expression of proinsulin in E. coli can be achieved by linking two or more proinsulin genes and expressing them directly or in conjuction with a small segment of the N-terminus of β-galactosidase.
Product Quality and Process
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Other amino acid sequence variants may occur post-translationally. At a low frequency, the leader sequence that directs the nascent protein to translocate into the endoplasmic reticulum may not be cleaved, producing a variant. Many proteins have lysine or arginine in their C-terminus. The basic amino acids in the C-terminus are liable to cleavage by carboxypeptidase. The cleavage of lysine at the C-terminus of the heavy chain of many IgG molecules by carboxypeptidase is often not complete, leaving some molecules with uncleaved C-terminus lysine.
Exploring the potential of microalgae cell factories for generation of biofuels
Published in Biofuels, 2023
Dixita Chettri, Ashwani Kumar Verma, Anil Kumar Verma
It has been reported that nitrogen deficiency positively affects the accumulation of TAG in certain microalgae, but the absence of nitrogen often inhibits cell growth. Therefore, an alternative strategy of expressing the cyanobacterial aryl acyl carrier protein (ACP) reductase in the chloroplasts of Cyanidioschyzon merolae (red alga) was investigated to increase TAG accumulation. The acyl-ACP reductase gene from Synechocystis sp. PCC 6803 was integrated into the genome of C. merolae under the influence of a catalase promoter with constitutive transcriptional activity. To direct the expressed enzyme into the chloroplast of the microalgae, a cleavable transit peptide of CmSecA [70] was fused to the N-terminus, while a 3 × His tag was used at the C-terminus to detect enzyme expression. The resulting stable transformant (AAR-3HA) showed up-regulation of the enzymes pyruvate dehydrogenase and acetyl-CoA carboxylase involved in FA synthesis in chloroplasts, while TCA-based consumption of acetyl-CoA was down-regulated. The results showed high accumulation of clear lipid droplets under nitrogen starvation and altered metabolic properties related to the expression of acyl-CoA reductase compared with wild-type [71].
Tailoring of recombinant FDH: effect of histidine tag location on solubility and catalytic properties of Chaetomium thermophilum formate dehydrogenase (CtFDH)
Published in Preparative Biochemistry & Biotechnology, 2019
Hacer Esen, Saadet Alpdağtaş, Mehmet Mervan Çakar, Barış Binay
In this study, we compared the solubilities, specific activities and kinetic parameters of two versions of CtFDH that differed in the position of His-tag at either N- or C- terminus. To incorporate of His-tag with the N-terminus or C-terminus of CtFDH has different effects on the catalytic properties of the enzyme. The results indicated that the N-terminal CtFDH has lower catalytic efficiency, turnover number, and specific activity than C-CtFDH in terms of both substrates. Therefore, it is necessary to keep the N-terminal region of CtFDH from interference such as His-tag fusion. When we evaluate the results of all the mentioned studies and this work, we cannot give general conclusions about the correct position of His-tag for FDHs in all living groups. Therefore, the effects of this label should be investigated individually for more FDHs originated from the same and different branch of living organisms to get a general inference.
The high expression of Aspergillus pseudoglaucus protease in Escherichia coli for hydrolysis of soy protein and milk protein
Published in Preparative Biochemistry and Biotechnology, 2018
Haiyan Liu, Rongzhen Zhang, Lihong Li, Lixian Zhou, Yan Xu
The effects of temperature on the expression of recombinant proteases in E. coli BL21 were evaluated at 20, 25, and 30 °C. SDS-PAGE analysis showed that App was highly expressed in E. coli BL21/pGEX-App at 20–30 °C, while the dominant expression band was not observed in E. coli BL21/pET-App (Figure 1a,c). The proApp was both expressed in E. coli BL21/pET-proApp and E. coli BL21/pGEX-proApp (Figure 1b,d). To eliminate the influence of N- or C- terminus histidine tag on protein expression, we constructed the N-terminus 7 × histidine tagged App and proApp on pGEX-6p-1. The results showed that N-terminus 7 × histidine tagged App and proApp were expressed at the similar level with C-terminus 7 × histidine tagged proteins (data not shown), suggesting the C- or N- terminus histidine tag did not affect the protein expression of App and proApp. The above results suggested that the propeptide is necessary for the successful expression of App on pET-28a (a)+ in E. coli. Wang et al. found the similar results that Rhizomumor miehei lipase was not expressed if the propeptide was removed artificially during the cloning process of lipase in E. coil.[23] The expression plasmid of pGEX-6p-1 also could help App to be expressed in E. coli BL21.