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Engineering Bactoblood for Oxygen Transport

Introduction

The primary function of human erythrocytes is to transport oxygen to the body's tissues and remove CO2. This is accomplished principly by high concentrations of the protein hemoglobin. However, functional expression of hemoglobin requires the coexpression of the small molecule (heme) that specifically binds oxygen, proteins that promote the expression, folding, and addition of heme to hemoglobin, and proteins that maintain the oxidation state of hemoglobin and prevent the accumulation of toxic oxidizing species in the cell. Bactoblood will similarly require these activities, so we designed a hierarchical genetic device that encoded this oxygen transport function. Our design contains a heme biosynthesis devices, a hemoglobin generation device, a chaperone device, and a detoxifying device. Additionally, we investigated alternatives to hemoglobin that may provide superior oxygen transport to Bactoblood than their human counterpart.

 

The Hemoglobin Generating Device

Background: Human Hemoglobin A and Methionine Aminopeptidase

The primary component needed for efficient oxygen transport in our system is human hemoglobin A. (HbA) HbA is a tetramer that consists of two different subunits, α2β2. We constructed a device that expresses both the alpha (HbA) and beta (HbB) subunits of human hemoglobin A, under the control of a T7 promoter. To cleave the extra methionine residue that is present when expressed in prokaryotic cells, we have also constructed a device which expresses the Map gene. Map encodes for methionine aminopeptidase, which will cleave the N-terminal methionine which is not normally present in eukaryotically expressed proteins. Thus, by expressing both of these devices, we achieve the expression of unmodified, fully functional adult human hemoglobin.

The Problem: Oxygen Binding Affinity and P50

The first problem to be addressed is the insufficiently low P50 of wild type human hemoglobin. The P50 is the partial pressure of oxygen needed for 50% saturation. A low P50 means that the oxygen affinity is too high, which inhibits the ability of the hemoglobin to deliver oxygen to the needed tissues. The P50 for wild type human hemoglobin is ~3.8 torr under normal physiological conditions, however this varies with temperature and pH.

Demonstrated by George J. Brewer's image to the left, an increase in P50 is the same as shifting the oxygen binding curve to the right. A shift to the right is shown to increase the oxygen transport, thus a higher P50 is desirable for efficient oxygen transport.

In human erythrocytes, the oxygen binding affinity is decreased by the presence of an allosteric modifiers, primarily 2,3-diphosphoglycerate (2,3-DPG), which forces the hemoglobin conformation into the lower affinity deoxy state, or the T-state. By pushing the hemoglobin into the T-state, 2,3-DPG is effectively pushing any bound oxygen out from the heme center. This effectively lowers the oxygen binding affinity of the hemoglobin and increases the P50. An increase of 0.4mM in DPG concentration decreases oxygen affinity by about 1.0 torr. Since the normal concentration of DPG in erythrocytes is ~5mM, this raises the P50 to ~16.3 torr.

DPG isn't the only allosteric modifier of hemoglobin, to a lesser extent, various ions bind to a region near the N-terminal end of the hemoglobin protein and act to increase the P50 as well. In recombinant hemoglobin expressed in E. coli, there is an extra methionine residue at the N-terminal region of expressed proteins. For the case of hemoglobin, that extra methionine acts to inhibit the allosteric effects of certain ions.

The Solution: Mutant Hemoglobin

In our system, we have chosen to use well studied mutants of human hemoglobin which have been engineered to be permanently in the deoxy T-state. First generation bactoblood will use a mutant which has two mutations applied to the beta subunit. The mutations are named Presbyterian (beta-Asn108Lys) and providence (beta-Lys82Asp). It has been reported that human hemoglobin with these two point mutations has a P50 in the range of ~44 torr, an acceptable P50 value for a good blood substitute.

 

Heme Biosynthesis Device

Heme is a prosthetic group to hemoglobin. Generally, heme consists of an iron atom surrounded by a porphyrin ring. Each hemoglobin molecule is capable of binding up to four heme groups. One of the most important functions of heme is to assist in the transportation of diatomic gases. In red blood cells, heme and hemoglobin are the components that allow the binding of oxygen. As a result of this interaction, red blood cells continuously deliver oxygen to the entire body.

 

 

The Chaperone Device

The alpha subunit is more prone to precipitation because an alpha-alpha dimer is insoluble under normal conditions. This can cause an excess of beta subunits and decreased functional hemoglobin output.

The solution in erythrocytes is to use a chaperone named alpha hemoglobin stabilizing protein (AHSP), which binds to single alpha subunits and facilitates the transfer of a beta subunit to the alpha. An alpha-beta dimer remains soluble. Our use of the bacterial chassis as a container that mimics an erythrocyte allows us to provide the same solution as in an erythrocyte. We include in our system the gene that encodes for human AHSP. In addition, we have also explored a fusion of di-alpha subunits with a glycine linker. This has been proven to give a higher soluble output by somehow stabilizing the alpha subunits and preventing precipitation.

 

Autoxidation Control

During the process of binding and unbinding oxygen, the four heme groups of the hemoglobin may spontaneously undergo autoxidation, ultimately causing the formation of damaging free radicals. This causes two problems. The first problem is that the free radicals can cause damage to cellular proteins, affecting their function. Another problem is that when autoxidation occurs, an electron is transferred from the Fe2+ iron center to the oxygen, creating a superoxide and leaving the heme with a Fe3+ metal center. The resulting hemoglobin is known as methemoglobin and it is unable to carry oxygen. The autoxidation occurs to a significant amount on the order of hours.
Erythrocytes remedy the problem of free radical accumulation and damage by containing the antioxidant enzymes catalase and superoxide dismutase. These enzymes catalyze the breakdown of superoxide into oxygen and H2O. Human erythrocytes have addressed the autoxidation problem by using the NADH dependent enzyme, cytochrome b5 reductase. The basic mechanism is shown here.

 

Hemoglobin Alternatives

We also investigated two alternatives to the hemoglobin part in our device: H-NOX and Myoglobin. Although the intrinsic oxygen-carrying ability of these proteins is different from Hemoglobin, variants of these proteins have been engineered with similar P50 values. These variants might allow Bactoblood to carry more oxygen than hemoglobin.

H-NOX is a heme-based sensor that is found in bacteria. H-NOX is able to bind Oxygen using a distal pocket tyrosine. For this gene I added the T7 promoter we created for this project, an RBS site, and lastly a Bca1092 terminator. When we assayed this part the results were inconclusive. The part was assembled correctly but the assay didn't show strong signs of expression.

The second gene we explored was Sperm Whale Myoglobin. Myoglobin is a monomeric protein that behaves as an intracellular oxygen storage site. Sperm whale myoglobin in particular is easily found in large amounts in the whale's muscle tissue. The construction of this part was very similar to that of the H-NOX composite part. It used the same promoter, terminator, and RBS. The assay for Myoglobin showed a bit more promise but couldn't conclusively show that Myoglobin was binding to oxygen.

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